Exhibit 10.15 CuraGen Corporation has omitted from this Exhibit 10.15 portions of the Agreement for which CuraGen Corporation has requested confidential treatment from the Securities and Exchange Commission. The portions of the Agreement for which...

Exhibit 10.15

CuraGen Corporation has omitted from this Exhibit 10.15 portions of the
Agreement for which CuraGen Corporation has requested confidential treatment
from the Securities and Exchange Commission. The portions of the Agreement for
which confidential treatment has been requested are marked with X's in brackets
and such confidential portions have been filed separately with the Securities
and Exchange Commission.

1. SUPERCEDES AWARD NOTICE dated ____________________

Restrictions previously imposed remain in effect unless specifically rescinded

--------------------------------------------------------------------------------
4. PLANT NO 5. ADMINISTRATIVE CODES
5 RO1 HGO1491-02 X7JP

Formerly:
--------------------------------------------------------------------------------
6. PROJECT PERIOD Mo., Day, Yr. Mo., Day, Yr.
05/01/96 04/30/99

From Through
--------------------------------------------------------------------------------
7. BUDGET PERIOD Mo., Day, Yr. Mo., Day, Yr.
05/01/97 04/30/98
From Through
--------------------------------------------------------------------------------

NATIONAL INSTITUTES OF HEALTH
NATIONAL HUMAN GENOME RESEARCH INSTITUTE

NOTICE OF GRANT AWARD
AUTHORIZATION (Legislation Regulation)

42 USC 241 42 CFR 52
RESEARCH
--------------------------------------------------------------------------------
8. TITLE OF PROJECT (Limit to 56 spaces)

AUTOMATED SEQUENCING SYSTEM FOR THE HUMAN GENOME PROJECT SRC (3)
--------------------------------------------------------------------------------
9. GRANTEE NAME AND ADDRESS

a. CURAGEN CORPORATION
000 X XXXX XX
x. XXXXXXXX, XX 00000

d.
--------------------------------------------------------------------------------
10. DIRECTOR OF PROJECT (PROGRAM DIRECTOR/PRINCIPAL INVESTIGATOR)
(LAST NAME FIRST AND ADDRESS)

WENT, XXXXXXX PHD
CURAGEN CORPORATION
LONG WHARF MARITIME CENTER
000 XXXX XXXXX XXXXX 00XX XX
XXX XXXXX, XXXXXXXXXXX 00000

--------------------------------------------------------------------------------
11. APPROVED BUDGET (Excludes PHS Direct Assurance)
--------------------------------------------------------------------------------
1 PHS Grant Funds Only
-----
[_] Total project costs including grant funds and all other I
Financial participation -----

(Select one and place NUMERAL in box.)
--------------------------------------------------------------------------------
a. Salaries and Wages . . . . $ [XXXXXX]

b. Fringe Benefits . . . . . $ [XXXXXX]

c. Total Personnel Costs . . . $ [XXXXXX]

d. Consultant Costs . . . . . . . . . . [XXXXXX]

e. Equipment. . . . . . . . . . . . . . [XXXXXX]

f. Supplies . . . . . . . . . . . . . . [XXXXXX]

g. Travel . . . . . . . . . . . . . . . [XXXXXX]

h. Patient Care -- Inpatient. . . . . . [XXXXXX]

i. -- Outpatient . . . . . [XXXXXX]

j. Alterations and Renovations. . . . . [XXXXXX]

k. Other. . . . . . . . . . . . . . . . [XXXXXX]

l. Consortium/Contractual Costs . . . . [XXXXXX]

m. Trainee Related Expenses . . . . . .

n. Trainee Stipends . . . . . . . . . . [XXXXXX]

o. Trainee Tuition and Fees . . . . . . [XXXXXX]

p. Trainee Travel . . . . . . . . . . . [XXXXXX]

-----------
q. TOTAL DIRECT COSTS $ [XXXXXX]
--------------------------------------------------------------------------------
r. INDIRECT COSTS Date ? of S&W TADC $ [XXXXXX]
--------------------------------------------------------------------------------
s. TOTAL APPROVED BUDGET $ [XXXXXX]
--------------------------------------------------------------------------------
t. SAIR Fee $ [XXXXXX]
--------------------------------------------------------------------------------
u. Federal Share. . . . . . . . . . . $ [XXXXXX]

v. Non-Federal Share. . . . . . . . . $
--------------------------------------------------------------------------------
12. AWARD COMPUTATION FOR FINANCIAL ASSISTANCE
--------------------------------------------------------------------------------
a. Amount of PHS Financial Assistance (from Item 11.v.). . . . $ [XXXXXX]

b. Less Unobligated Balance From Prior Budget Periods. . . . . $ [XXXXXX]

c. Less Cumulative Prior Award(s) This Budget Period . . . . . $ [XXXXXX]

-------------
d. AMOUNT OF FINANCIAL ASSISTANCE THIS ACTION. . . . . . . . . $ [XXXXXX]
--------------------------------------------------------------------------------
13. RECOMMENDED FUTURE SUPPORT (SUBJECT TO THE AVAILABILITY OF FUNDS AND
SATISFACTORY PROGRESS OF THE PROJECT-
--------------------------------------------------------------------------------
YEAR TOTAL DIRECT COSTS/STIPENDS YEAR TOTAL DIRECT COSTS/STIPENDS
--------------------------------------------------------------------------------
a. 03 [XXXXXX] d.

b. e.

c. f.
--------------------------------------------------------------------------------
14. APPROVED DIRECT ASSISTANCE BUDGET (IN LIEU OF CASH)

a. Amount of PHS Direct Assistance . . . . . . . . . . . $

b. Less Unobligated Balance From Prior Budget Periods. . $

c. Less Cumulative Prior Award(s) This Budget Period . . $

-------------------
d. AMOUNT OF DIRECT ASSISTANCE THIS ACTION . . . . . . . $
--------------------------------------------------------------------------------
15. PROGRAM INCOME SUBJECT TO 45 CFR PART 74, SUBPART F. OR 45 CFR 92.25.
SHALL BE USED IN ACCORD WITH ONE OF THE FOLLOWING ALTERNATIVES:
(Select One and Place LETTER in box.)

a. DEDUCTION

b. ADDITIONAL COSTS
-----
c. MATCHING A
-----
d. OTHER RESEARCH (Add Deduct Option)

e. OTHER (See REMARKS)
--------------------------------------------------------------------------------
16. THIS AWARD IS BASED ON AN APPLICATION SUBMITTED TO AND AS APPROVED BY THE
PHS ON THE ABOVE TITLED PROJECT AND IS SUBJECT TO THE TERMS AND CONDITIONS
INCORPORATED EITHER DIRECTLY OR BY REFERENCE IN THE FOLLOWING:

a. The grant program legislation cited above.

b. The grant program regulation cited above.

c. This award notice including terms and conditions, if any, noted below
under REMARKS.

d. PHS Grants Policy Statement including addenda in effect as of the
beginning date of the budget period.

e. 45 CFR Part 74 or 45 CFR Part 92 at applicable.

In the event there are conflicting or otherwise inconsistent policies
applicable to the grant, the above order of precedence shall prevail.
Acceptance of the grant terms and conditions is acknowledged by the grantee
when funds are drawn or otherwise obtained from the grant payment system.
--------------------------------------------------------------------------------
REMARKS: (Other Terms and Conditions Attached - [X] Yes [_] No)

GRANT SPECIALIST: XXXXX XXXX 000-000-0000
PROGRAM OFFICIAL: XX. X. XXXXXXXX 000-000-0000

* Indirect costs were calculated as indicated in Term and Condition #11.
** RECOMMENDED FUTURE SUPPORT REFLECTS TOTAL COSTS.

THIS GRANT IS EXCLUDED FROM STREAMLINED NONCOMPETING AWARD PROCEDURES (SNAP).
THIS GRANT IS INCLUDED UNDER EXPANDED AUTHORITIES.
--------------------------------------------------------------------------------
PHS GRANTS MANAGEMENT OFFICER: (Signature)

/s/ Xxxx X. Xxxxxx
--------------------------------------------------------------------------------
(Name/Typed/Print)

XXXX X. XXXXXX
--------------------------------------------------------------------------------
(Title)

GRANTS MANAGEMENT OFFICER
--------------------------------------------------------------------------------
17. OBJ. CLASS 18. CRS - EIN 19. LIST NO.
41 4E 1061331400A1
--------------------------------------------------------------------------------
FY CAN DOCUMENT NO. ADMINISTRATIVE CODE
20.a. b. c.

21.a. 97 8427201 b. R1HG01491A c.

22.a. 970423 1710 b. c.
--------------------------------------------------------------------------------
AMT ACTION FIN ASST. AMT ACTION DIR ASST.
d. e.

d. e.

d. e.
--------------------------------------------------------------------------------
PHS 5152-5 (Rev. 7/94) (Note: See reverse for payment information.) 13G

NOTICE OF GRANT AWARD

Page 2
5 R01 HG01491-02
WENT, XXXXXXX X., PH.D.

1. The terms and conditions include the requirements of the Omnibus
Consolidated FY 1997 Appropriations Act (P. L. 104-208) as explained in the NIH
Guide for Grants and Contracts, Volume 26, Number 4, February 7, 1997.

2. The grantee institution may not provide any funds from this award to any
institution with which it has a research subcontract/consortium agreement until
the grantee has ensured that the third party institution has complied with all
applicable certifications and assurances as specified in the PHS Grants Policy
Statement. The grantee is responsible for retaining documentation to this
effect.

3. Applicant organization must comply with the Public Health Service (PHS)
policy relating to distribution of unique research resources produced with PHS
funding (NIH Guide for Grants and Contracts, Vol. 25, no. 23, July 12, 1996).
Grantee should also comply with the NIH-DOE guidelines for access to mapping and
sequencing data and materials resources, adopted by the NIH-DOE joint
subcommittee on December 7, 1992.

4. Allowable costs of activities conducted by for-profit organizations will be
determined by applying the costs principles of Contracts with Commercial
Organizations set forth in 48 CFR 31.2. However, independent research and
development costs (including the indirect costs allocable to them) are
unallowable.

5. Normally, the awardee organization retains the principal worldwide patent
rights to any invention developed with United States government support. Under
Title 37 Code of Federal Regulations Part 401, the Government receives a
royalty-free license for its use, reserves the right to require the patent
holder to license others in certain circumstances, and requires that anyone
exclusively licensed to sell the invention in the

NOTICE OF GRANT AWARD

Page 3
5 R01 HG01491-02
WENT, XXXXXXX X., PH.D.

United States must normally manufacture it substantially in the United States.
To the extent authorized by Title 00 Xxxxxx Xxxxxx Code Section 205, the
Government will not make public any information disclosing a
Government-supported invention for a 4-year period to allow the awardee
organization a reasonable time to file a patent application, nor will the
Government release any information that is part of that application.

6. Two months after disclosure, any invention developed under this award should
be reported to the Office for Policy on Extramural Research Administration
(OPERA), 0000 Xxxxxxxxx Xxxxx, XXX 0000, Xxxxxxxx, XX 00000-0000, Attn: Xxx
Xxxxx. Phone (000) 000-0000, for disposition of patent rights in accordance with
45 CFR, Parts 6 and 8. Disposition rights on inventions made by small businesses
are subject to Chapter 18 of Title 35 U.S. Code.

7. Prior approval to implement budgetary and programmatic changes, where
required by PHS Policy, must be obtained in writing from the Grants Management
contact shown on this Notice of Grant Award.

8. The indirect cost portion of the commitments is calculated using the current
negotiated indirect cost (IC) rates, as follows:

03 Year

Direct Costs: $ [XXXXXX]
IC Calculation:
$ [XXXXXX] (fringe)
$ [XXXXXX]
Total indirect costs: [XXXXXX]
Total costs: $ [XXXXXX]

9. Effective with the August 25, 1994 issuance of 45 CFR Part 74, certain
expanded authorities were extended to all research grants and cooperative
agreements. Under this grant award, the carryover authority is not authorized.

NOTICE OF GRANT AWARD
(Additional Remarks)

Page 5

If you need assistance from National Center for Human Genome Research during the
course of this grant, please contact the grants management and program staff
whole telephone numbers are shown on the Notice of Grant Award. These
individuals work closely with one another through all phases of each project to
facilitate the award and the administration of the grant. Their functions are
defined as follows:

GRANTS MANAGEMENT CONTACT: The Grants Management Specialist, whose name and
telephone number are indicated in the "Remarks" section of the Notice of Grant
Award, is responsible for all business management matters associated with the
review, negotiation, award, and administration of grants. The Grants Management
Specialist serves as the focal point for receiving and responding to all
questions and correspondence related correspondence giving or denying any prior
approval required by Public Health Service policy or special Terms of Award,
transfer of the grant to another institution, a change in the period of support,
or any actions which commit, or may result in committing the NCHGR to a change
in the amount of funding.

PROGRAM CONTACT: The Health Scientist Administrator, or program official, whose
name and telephone number also are indicated in the "Remarks" section of the
Notice of Grant Award, is responsible for all scientific and technical matters
dealing with the administration of the grant. The Health Scientist Administrator
reviews and monitors scientific progress of the project and provides advice and
assistance relative to all technical problems to ensure that the scientific
objectives of the research program can be pursued effectively and successfully.
All questions or correspondence dealing with research progress, changes in
research direction, unique scientific opportunities, or any other scientific
need should be addressed to the Health Scientist Administrator.

PRIOR APPROVAL: Requests which require the prior approval of the NCHGR must be
submitted in writing to the Grants Management Officer. All requests should
reference the complete grant number, e.g., 1 R01 HG12345-01, and must be signed
by the authorized official of the business office of the grantee institution and
by the principal investigator.

------------------------------------------------------------------------------------------------------------------------
DEPARTMENT OF HEALTH AND HUMAN SERVICES LEAVE BLANK FOR PHS USE ONLY.
-------------------------------------------------------------------------
PUBLIC HEALTH SERVICE Type Activity Number
-------------------------------------------------------------------------
GRANT APPLICATION Review Group Formerly
-------------------------------------------------------------------------
Follow Instruction carefully. Type in the Council/Board (Month, Year) Date Received
unshaded areas only. Type density must be
10 cpi
------------------------------------------------------------------------------------------------------------------------
1. TITLE OF PROJECT (Do not exceed 55 typewritten spaces.)
Automated Sequencing System for the Human Genome Project
------------------------------------------------------------------------------------------------------------------------
2a. RESPONSE TO SPECIFIC REQUEST FOR APPLICATIONS OR PROGRAM ANNOUNCEMENT [_] NO [X] YES (if "YES", state number)
Number HG-95-004 Title: Improved Electrophoretic DNA Sequencing Technologies
------------------------------------------------------------------------------------------------------------------------
2b. TYPE OF GRANT PROGRAM R01 3. PRINCIPAL INVESTIGATOR/PROGRAM DIRECTOR
------------------------------------------------------------------------------------------------------------------------
3a. NAME (Last, first, middle) 3b. DEGREE(S) 3c. SOCIAL SECURITY NO.
Went, Xxxxxxx T. B.S. Ph.D. ###-##-####
------------------------------------------------------------------------------------------------------------------------
3d. POSITION TITLE 3e. XXXXXXX XXXXXXX (Xxxxxx, xxxx, xxxxx, zip code)
VICE PRESIDENT
--------------------------------------------------
3f. DEPARTMENT, SERVICE, LABORATORY, OR EQUIVALENT
CuraGen Corporation 000 Xxxx Xxxx Xxxxxx
-------------------------------------------------- Branford, CT 06405
3g. MAJOR SUBDIVISION
None
-------------------------------------------------
3h. TELEPHONE AND FAX (Area code, number and extension
TEL: (203) 481-1104 ext. 31
FAX: (000) 000-0000 --------------------------------------------------------------------
BITNET/INTERNET ADDRESS xxxxx@xxxxxxx.xxx
------------------------------------------------------------------------------------------------------------------------
4. HUMAN SUBJECTS IRB 5. VERTEBRATE ANIMALS if "YES" 5b. Animal Welfare
If "Yes" approval 4b. Assurance of IACUE approval date assurance no.
----- 4a. ---- exemption no. or date compliance no. ----- 5a. -----
X NO YES X NO YES
------------------------------------------------------------------------------------------------------------------------
6. DATES OF ENTIRE PROPOSED PROJECT 7. COSTS REQUESTED FOR INITIAL 8. COSTS REQUESTED FOR ENTIRE
PERIOD BUDGET PERIOD PROPOSED PROJECT PERIOD

From(MMDDYY) Through(MMDDYY) 7a. Direct Costs($) 7b. Total Costs($) 8a. Direct Costs($) 8b. Total Costs
04/01/96 03/31/99 $ [XXXXXX] $ [XXXXXX] $ [XXXXXX] $ [XXXXXX]
------------------------------------------------------------------------------------------------------------------------
9. PERFORMANCE SITES (Organizations and addresses) 10. INVENTIONS AND PATENTS (Competing continuation application only)

---- ---- If ---- previously ---- not previously
CuraGen Corporation Cornell University NO YES "YES" reported reported
000 Xxxx Xxxx Xxxxxx College of Engineering ---------------------------------------------------------------
Xxxxxxxx, XX 00000 Xxxxx Xxxx 11. NAME OF APPLICANT ORGANIZATION
Xxxxxx, XX 00000-0000
CuraGen Corporation
ADDRESS 000 Xxxx Xxxx Xxxxxx
Suane BioSciences, Inc. The Xxxxxxxxx Institute for Xxxxxxxx, XX 00000
0000 Xxxxx Xxx Xxxxxxxxxx Xxxxxxxx
Xxxxxxxx, XX 00000 Nine Xxxxxxxxx Xxxxxx
Xxxxxxxxx, XX 00000-0000
------------------------------------------------------------------------------------------------------------------------
12. TYPE OF ORGANIZATION 13. ENTITY IDENTIFICATION NUMBER Congressional District
[_] Public specify [_] Federal [_] State [_] Local 00-0000000 #3
---------------------------------------------------------------
[_] Private nonprofit 14. BIOMEDICAL RESEARCH SUPPORT GRANT CREDIT
[X] For profit (General) [_] (Small Buiness) Code: Identification:
------------------------------------------------------------------------------------------------------------------------
15. NAME OF ADMINISTRATIVE OFFICIAL TO BE NOTIFIED IF 16. NAME OF OFFICIAL SIGNING FOR APPLICANT ORGANIZATION
AWARD IS MADE
Xxxxxxx X. Went, Ph.D. Xxxxxxx X. Went, Ph.D.
TELEPHONE (000) 000-0000, Ext 31 TELEPHONE (000) 000-0000, Ext 31
FAX (000) 000-0000 FAX (000) 000-0000
TITLE Vice President TITLE Vice President
ADDRESS CuraGen Corporation ADDRESS CuraGen Corporation
000 Xxxx Xxxx Xxxxxx 000 Xxxx Xxxx Xxxxxx
Xxxxxxxx, XX 00000 Xxxxxxxx, XX 00000

BITNET/INTERNET ADDRESS xxxxx@xxxxxxx.xxx BITNET/INTERNET ADDRESS xxxxx@xxxxxxx.xxx
------------------------------------------------------------------------------------------------------------------------
17. PRINCIPAL INVESTIGATOR/PROGRAM DIRECTOR SIGNATURE OF PERSON NAMED IN 3a. DATE
ASSURANCE: I agree to accept responsibility (In ink. "Per" signature
for the scientific conduct of the project not acceptable.)
and to provide and to provide the required /s/ Xxxxxxx Went 8/25/95
progress reports if a grant is awarded as a
result of this application. Will ful provision
of false information is a criminal offense
(U.S. Code, Title 18, Section 1001). I am aware
that any false, fictitious or fraudulent
statement may, in addition to other remedies
available to the Government, subject me to
civil penalties under the Program Fraud
Civil Remedies Act of 1986 (45 CFR 79).
------------------------------------------------------------------------------------------------------------------------
18. CERTIFICATION AND ACCEPTANCE: I certify that the SIGNATURE OF PERSON NAMED IN 15. DATE
statements herein are true and complete to the (In ink. "Per" signature
best of my knowledge, and accept the obligation to not acceptable.)
comply with Public Health Services terms and /s/ Xxxxxxx Went 8/25/95
conditions if a grant is awarded as the result of
this application. A willfully false certification
is a criminal offense (U.S. Code, Title 18, Section
1001). I am aware that any false, fictitious, or
fraudulent statement may, in addition to other
remedies available to the Government, subject me
to civil penalties under the Program Fraud Civil
Remedies Act of 1986 (45 CFR 79).
------------------------------------------------------------------------------------------------------------------------

[Confidential Treatment Requested]

BB
Principal Investigator/Program Director (Last, first, middle):
Went, Xxxxxxx X.
----------------
--------------------------------------------------------------------------------

DESCRIPTION: State the application's broad, long-term objectives and specific
aims, making reference to the health relatedness of the project. Describe
concisely the research design and methods for achieving these goals. Avoid
summaries of past accomplishments and the use of the first person. This abstract
is meant to serve as a succinct and accurate description of the proposed work
when separated from the application. DO NOT EXCEED THE SPACE PROVIDED.
--------------------------------------------------------------------------------
The genetic information encoded in the human genome specifies all life processes
from conception to death, and contains within it the origin of over 3500 known
genetic diseases. It is necessary to substantially reduce the cost and
dramatically raise the speed of producing accurate DNA sequences in order to
determine the sequence of the 3 billion basepairs of DNA that compose the human
genome. This proposal describes a three-year project to develop, integrate and
distribute into parallel sequencing projects a fluorescent sequencing instrument
and software system capable of meeting these logistical and cost demands. This
approach allows for the identification and removal of the major bottlenecks
encountered in producing a fully automated system, without duplication of
mapping, sample processing or informatics efforts ongoing at other HGP
facilities. Current bottlenecks arise primarily from inefficiencies in workflow
and data analysis. Specifically, we will develop and integrate into existing
centers the following: 8-dye bi-directional sequencing methods; solid phase
loading of an advanced electrophoresis instrument; high-throughput and low cost
ready-to-run disposable microeplicated separation sub-systems; sensitive
detection optics; and advanced base-calling software. These advances will enable
the completion of fully automated systems including all the steps necessary to
proceed from a physical map to a finished sequence with a quantifiable measure
of confidence. Full process automation coupled with automated data analysis and
reduced materials cost will enable a 60-fold increase in throughput with the
required 10-fold decrease in cost relative to systems based on currently
available technology. This project is coordinated by CuraGen Corporation and
builds on the automation, data analysis, sequencing instrumentation, and
fabrication experience of collaborators at Soane BioSciences, MIT and Cornell.
The final product of our efforts will be tested and ready for wide-spread
distribution for use in sequencing the human genome.
--------------------------------------------------------------------------------
PERSONNEL ENGAGED ON PROJECT, INCLUDING CONSULTANTS/COLLABORATORS. Use
continuation pages as needed for ?????? required information in the format shown
below on all individuals participating in the project.
--------------------------------------------------------------------------------

Name Xxxxxxx X. Went Degree(s) Ph.D. Social Security ####-##-####
------------------------------------------------------------ ------------- ----------------
Position Title Vice President D.O.B. 7/1/63 Role on Project Prin. Investig.
---------------------------------------------------- ----------------- -----------------
Organization CuraGen Corporation Department
------------------------------------------------------------------------------ -----------------
Name Xxxxxxxxx X. Xxxxxxxx Degree(s) Ph.D. Social Security ####-##-####
------------------------------------------------------------ ------------- ----------------
Position Title Chief Executeve Officer D.O.B. 4/28/62 Role on Project Scientist
---------------------------------------------------- ----------------- -----------------
Organization CuraGen Corporation Department
------------------------------------------------------------------------------ -----------------
Name Xxxxxxx X. XxXxxxx Degree(s) Ph.D. Social Security ####-##-####
------------------------------------------------------------ ------------- ----------------
Position Title Senior Research Scientist D.O.B. 3/3/62 Role on Project Scientist
---------------------------------------------------- ----------------- -----------------
Organization CuraGen Corporation Department
------------------------------------------------------------------------------ -----------------
Name Xxxxxxx X. Xxxxxxx Degree(s) Ph.D. Social Security ####-##-####
------------------------------------------------------------ ------------- ----------------
Position Title Senior Research Scientist D.O.B. 8/11/67 Role on Project Scientist
---------------------------------------------------- ----------------- -----------------
Organization CuraGen Corporation Department
------------------------------------------------------------------------------ -----------------
Name Xxxxx X. Xxxxxxx Degree(s) Ph.D. Social Security ####-##-####
------------------------------------------------------------ ----------------- -----------------
Position Title Senior Research Engineer D.O.B. 6/1/63 Role on Project Engineer
---------------------------------------------------- ----------------- -----------------
Organization CuraGen Corporation Department
------------------------------------------------------------------------------ -----------------
Name Xxxx X. Xxxxxxx Degree(s) M.S. Social Security ####-##-####
------------------------------------------------------------ ------------- ----------------
Position Title Senior Research Engineer D.O.B. 3/6/65 Role on Project Engineer
---------------------------------------------------- ----------------- -----------------
Organization CuraGen Corporation Department
------------------------------------------------------------------------------ --------------------

BB
Principal Investigator/Program Director (Last, first, middle):
Went, Xxxxxxx X.
----------------
--------------------------------------------------------------------------------

Name Xxxxxxxxxx Xxxxxxxxxxxxx Degree(s) Ph.D. Social Security ####-##-####
------------------------------------------------------------ ------------- ------------------

Position Title Director of Bioinformatics D.O.B. 04/12/61 Role on Project Scientist
---------------------------------------------------- ---------------- ------------------

Organization CuraGen Corporation Department
----------------------------------------------------------------------------- ------------------

Name Xxxxxx X. Xxxxxxx Degree(s) B.S. Social Security ####-##-####
------------------------------------------------------------ ------------- ------------------

Position Title Director of Software Development D.O.B. 3/25/59 Role on Project Scientist
---------------------------------------------------- ---------------- ------------------

Organization CuraGen Corporation Department
----------------------------------------------------------------------------- ------------------

Name Xxxxxx X. Xxxxxxxxxx Degree(s) B.S. Social Security ####-##-####
------------------------------------------------------------ ------------- ------------------

Position Title Technician D.O.B. 12/03/66 Role on Project
---------------------------------------------------- ---------------- ------------------

Organization CuraGen Corporation Department
----------------------------------------------------------------------------- ------------------

Name To Be Hired Degree(s) Social Security #
------------------------------------------------------------ ------------- ------------------

Position Title Fabrication D.O.B. Role on Project
---------------------------------------------------- ---------------- ------------------

Organization CuraGen Corporation Department
----------------------------------------------------------------------------- ------------------

Name To Be Hired Degree(s) Social Security #
------------------------------------------------------------ ------------- ------------------

Position Title Programmer D.O.B. Role on Project
---------------------------------------------------- ---------------- ------------------

Organization CuraGen Corporation Department
----------------------------------------------------------------------------- ------------------

Name Xxxxxx Xxxxxxxxx Degree(s) Ph.D. Social Security ####-##-####
------------------------------------------------------------ ------------- ------------------

Position Title Professor D.O.B. 9/21/52 Role on Project
---------------------------------------------------- ---------------- ------------------

Organization Cornell University Department
----------------------------------------------------------------------------- ------------------

Name Xxxxxx Xxxxxx Degree(s) Ph.D. Social Security ####-##-####
------------------------------------------------------------ ------------- ------------------

Position Title Research Associate D.O.B. 12/11/50 Role on Project Research Assoc.
---------------------------------------------------- ---------------- ------------------

Organization Cornell University Department
----------------------------------------------------------------------------- ------------------

Name To Be Hired Degree(s) Social Security #
------------------------------------------------------------ ------------- ------------------

Position Title Graduate Student D.O.B. Role on Project
---------------------------------------------------- ---------------- ------------------

Organization Cornell University Department
----------------------------------------------------------------------------- ------------------

Name Xxxxxxx X. Xxxxxx Degree(s) Ph.D. Social Security ####-##-####
------------------------------------------------------------ ------------- ------------------

Position Title Vice President of Research D.O.B. 09/01/63 Role on Project Prin. Investig.
---------------------------------------------------- ---------------- ------------------

Organization Soane BioSciences, Inc. Department
----------------------------------------------------------------------------- ------------------

Name Xxxxx X. Xxxxx Degree(s) Ph.D. Social Security ####-##-####
------------------------------------------------------------ ------------- ------------------

Position Title Chairman, Scientific Advisory Board D.O.B. 08/22/51 Role on Project Scientist
---------------------------------------------------- ---------------- ------------------

Organization Soane Technologies, Inc. Department
----------------------------------------------------------------------------- ------------------

Name Xxxxxxx Xxxxxx Amigo Degree(s) Ph.D. Social Security ####-##-####
------------------------------------------------------------ ------------- ------------------

Position Title Senior Scientist D.O.B. 10/01/56 Role on Project Scientist
---------------------------------------------------- ---------------- ------------------

Organization Soane BioSciencies, Inc. Department
----------------------------------------------------------------------------- ------------------

Name Xxxxxxxxx X. Xxxxx Degree(s) Ph.D. Social Security ####-##-####
------------------------------------------------------------ ------------- ------------------

Position Title Senior Research Engineer D.O.B. 04/25/67 Role on Project Engineer
---------------------------------------------------- ---------------- ------------------

Organization Soane BioSciences, Inc. Department
----------------------------------------------------------------------------- ------------------

Name Xxxxxx Xxxx Degree(s) B.S. Social Security ####-##-####
------------------------------------------------------------ ------------- ------------------

Position Title Research Biochemist D.O.B. 03/02/70 Role on Project Biochemist
---------------------------------------------------- ---------------- ------------------

Organization Soane BioSciences, Inc. Department
---------------------------------------------------- ------------- ------------------

Name Xxx Xxx Degree(s) Ph.D. Social Security ####-##-####
------------------------------------------------------------ ------------- ------------------

Position Title Postdoctoral D.O.B. 08/24/62 Role on Project Scientist
---------------------------------------------------- ---------------- ------------------

Organization Soane BioSciencies, Inc. Department
----------------------------------------------------------------------------- ------------------

Name Xxxxx Xxxxxxxxx Degree(s) B.S. Social Security ####-##-####
------------------------------------------------------------ ------------- ------------------

Position Title Designer/Machinist D.O.B. 04/30/33 Role on Project Machinist
---------------------------------------------------- ---------------- ------------------

Organization Soane BioSciences, Inc. Department
----------------------------------------------------------------------------- ------------------

Name To Be Determined Degree(s) Social Security #
------------------------------------------------------------ ------------- ------------------

Position Title Polymer Engineer D.O.B. Role on Project
---------------------------------------------------- ---------------- ------------------

Organization Soane BioSciences, Inc. Department
----------------------------------------------------------------------------- ------------------

Name Xxxxxx X. Xxxxxxx Degree(s) Ph.D. Social Security ####-##-####
------------------------------------------------------------ ------------- ------------------

Position Title Research Scientist D.O.B. 03-07-60 Role on Project Prin. Investig.
---------------------------------------------------- ---------------- ------------------

Organization The Xxxxxxxxx Institute Department
----------------------------------------------------------------------------- ------------------

Name Lincoln X. Xxxxx Degree(s) M.D.,Ph.D. Social Security ####-##-####
------------------------------------------------------------ ------------- ------------------

Position Title Director of Informatics Core D.O.B. 01/13/60 Role on Project Scientist
---------------------------------------------------- ---------------- ------------------

Organization The Xxxxxxxxx Institute of Biomedical Research, MIT Department
----------------------------------------------------------------------------- ------------------

Name Xxxxx Xxxx Degree(s) B.S. Social Security ####-##-####
------------------------------------------------------------ ------------- ------------------

Position Title Technical Assistant D.O.B. 12/13/71 Role on Project Technican
---------------------------------------------------- ---------------- ------------------

Organization The Xxxxxxxxx Institute of Biomedical Research, MIT Department
----------------------------------------------------------------------------- ------------------

Name Xxxxxxx Xxx Degree(s) B.S. Social Security ####-##-####
------------------------------------------------------------ ------------- ------------------

Position Title Research Specialist D.O.B. 12/23/74 Role on Project Research
---------------------------------------------------- ---------------- ------------------

Organization The Xxxxxxxxx Institute of Biomedical Research, MIT Department
----------------------------------------------------------------------------- ------------------

Principal Investigator/Program Director (Last, first, middle):
Went, Xxxxxxx X.
---------------
-------------------------------------------------------------------------------

RESEARCH GRANT
TABLE OF CONTENTS

PAGE NUMBERS
------------

Face Page................................................................................. 1
Description and Personnel................................................................. 2-3
Table of Contents......................................................................... 4
Detailed Budget for Initial Budget Period................................................. 5
Budget for Entire Proposed Project Period................................................. 6-9
Budgets Pertaining to Consortium/Contractual Arrangements................................. 10-21
Biographical Sketch-Principal Investigator/Program Director (Not to exceed two pages)..... 22-23
Other Biographical Sketches (Not to exceed two pages for each)............................ 24-49
Other Support............................................................................. 50-74
Resources and Environment................................................................. 75-78

Research Plan

Introduction to Revised Application (Not to exceed three pages).......................... _______
Introduction to Supplemental Application (Not to exceed one page)......................... _______
1. Specific Aims.................................................................... 79-80
2. Background and Significance...................................................... 80-84
3. Progress Report/Preliminary Studies (Not to exceed 25 pages*).................... 85-92
4. Research Design and Methods...................................................... 92-103
5. Human Subjects................................................................... _______
6. Vertebrate Animals............................................................... _______
7. Consultants/Collaborators........................................................ 104-107
8. Consortium/Contractual Arrangements.............................................. 108-111
9. Literature Cited (Not to exceed six pages)....................................... 112-114
Checklist................................................................................. 115-116
*Type density and size must conform to limits provided in Specific Instructions on page 10.

Appendix (Five collated sets. No page numbering necessary for Appendix)
Number of publications and manuscripts accepted or submitted for
publication (Not to exceed 10):
Other items (list):
[X] Check if Appendix is
Included

Appendix 1: Color Copies of Figures
Appendix 2: Detailed Cost Analysis

DD Principal Investigator/Program Director (Last, first, middle):
Went, Xxxxxxx X.
----------------
--------------------------------------------------------------------------------

------------------------------------------------------------------------------------------------------------------------------
DETAILED BUDGET FOR INITIAL BUDGET PERIOD FROM FROM THROUGH
DIRECT COSTS ONLY (CuraGen Corporation) 04/01/96 03/31/97
------------------------------------------------------------------------------------------------------------------------------
PERSONNEL (Applicant Organization Only) DOLLAR AMOUNT REQUESTED (omit cents)
------------------------------------------ INST. -----------------------------------------------
ROLE ON TYPE APPT. % EFFORT BASE SALARY FRINGE
NAME PROJECT (months) ON PROJ. SALARY REQUESTED BENEFITS TOTALS
------------------------------------------------------------------------------------------------------------------------------

------------------------------------------------------------------------------------------------------------------------------
Principal
Xxxxxxx X. Went Investigator 12 [XXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX
------------------------------------------------------------------------------------------------------------------------------
Xxxxxxxx X. Xxxxxxxx CSO/CEO 12 XXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX
------------------------------------------------------------------------------------------------------------------------------
Senior
Xxxxxxx X. XxXxxxx Scientist 12 XXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX
------------------------------------------------------------------------------------------------------------------------------
Senior
Xxxxxxx X. Xxxxxxx Xxxxxxxxx 00 XXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX
------------------------------------------------------------------------------------------------------------------------------
Senior
Xxxxx X. Xxxxxxx Engineer 12 XXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX
------------------------------------------------------------------------------------------------------------------------------
Senior
Xxxx X. Xxxxxxx Engineer 12 XXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX
------------------------------------------------------------------------------------------------------------------------------
Director of
Xxxxxxxxxx Xxxxxxxxxxxxx Bioinformatics 12 XXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX
------------------------------------------------------------------------------------------------------------------------------
Director of
Xxxxxx X. Xxxxxxx Software Dev 12 XXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX
------------------------------------------------------------------------------------------------------------------------------
Xxxxxx X. Xxxxxxxxxx Technician 12 XXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX
------------------------------------------------------------------------------------------------------------------------------
To be hired Fabrication 12 XXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX
------------------------------------------------------------------------------------------------------------------------------
To be hired Programmer 12 XXX XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX]
------------------------------------------------------------------------------------------------------------------------------
-------------------------------------------------------------------------------===============================================
SUBTOTALS----------------------------------------- [XXXXXXXX XXXXXXXX XXXXXXXX]
-------------------------------------------------------------------------------===============================================

CONSULTANT COSTS
[XX]
------------------------------------------------------------------------------------------------------------------------------
[XXXXXXXXXXXXXXXXXX]
[XXXXXXXX] [XXXXXX] Optical components [XXXXXX]
[XXXXXXXX] [XXXXXX] Software [XXXXXX]
Lab equipment [XXXXXX] Support hardware [XXXXXX]
Power supplies [XXXXXX] [XXXXXX]
------------------------------------------------------------------------------------------------------------------------------
SUPPLIES (Itemize by category)
Substrates for fabrication [XXXXXX] Plastic disposables [XXXXXX]
Other fabrication materials/reagents [XXXXXX] Test reagents/chemicals [XXXXXX]
Gel box materials [XXXXXX] Fluorescent labelling [XXXXXX]
DNA sequence supplies [XXXXXX] [XXXXXX]
------------------------------------------------------------------------------------------------------------------------------
TRAVEL
Sixteen trips per year to conferences and MIT [XXXXXX]
Two trips per year for P.I. for semi-annual project management [XXXXXX] [XXXXXX]
------------------------------------------------------------------------------------------------------------------------------
PATIENT CARE COSTS INPATIENT [XXXXXX]
---------------------------------------------------------------------------------------------------
OUTPATIENT [XXXXXX]
------------------------------------------------------------------------------------------------------------------------------
ALTERATIONS AND RENOVATIONS (Itemize by category)
[XXXXXX]
------------------------------------------------------------------------------------------------------------------------------
OTHER EXPENSES (Itemize by category)
Machining/Engineering [XXXXXXXXXXXXXXXXXXXXX] [XXXXXX]
Fabrication - NNF [XXXXXXXXXXXXXXXXXXXXX] [XXXXXX]
Publication Costs [XXXXXX] [XXXXXX]
-------------------------------------------------------------------------------------------------------------=================
SUBTOTAL DIRECT COSTS FOR INITIAL BUDGET PERIOD [XXXXXX]
-------------------------------------------------------------------------------------------------------------=================
CONSORTIUM/CONTRACTUAL COSTS
DIRECT COSTS [XXXXXX]
---------------------------------------------------------- TOTAL---- [XXXXXX]
INDIRECT COSTS [XXXXXX]
-------------------------------------------------------------------------------------------------------------=================
TOTAL DIRECT COSTS FOR INITIAL BUDGET PERIOD (Item 7a, Face Page)---- [XXXXXX]
==============================================================================================================================

[Confidential Treatment Requested]

Principal Investigator/Program Director (Last, first, middle)
Went, Xxxxxxx X.
----------------

------------------------------------------------------------------------------------------------------------------
BUDGET FOR ENTIRE PROPOSED PROJECT PERIOD
DIRECT COSTS ONLY (CuraGen Corporation)

------------------------------------------------------------------------------------------------------------------
BUDGET CATEGORY INITIAL BUDGET ADDITIONAL YEARS OF SUPPORT REQUESTED
PERIOD --------------------------------------------------------------
TOTALS (from page 4) 2nd 3rd 4th 5th
------------------------------------------------------------------------------------------------------------------

PERSONNEL:
Salary & fringe benefits
Applicant organization only [XXXXXXXX XXXXXXXX XXXXXXXX
------------------------------------------------------------------------------------------------------------------
CONSULTANT COSTS XXXXXXXX XXXXXXXX XXXXXXXX
------------------------------------------------------------------------------------------------------------------
EQUIPMENT XXXXXXXX XXXXXXXX XXXXXXXX
------------------------------------------------------------------------------------------------------------------
SUPPLIES XXXXXXXX XXXXXXXX XXXXXXXX
------------------------------------------------------------------------------------------------------------------
TRAVEL XXXXXXXX XXXXXXXX XXXXXXXX
------------------------------------------------------------------------------------------------------------------
PATIENT INPATIENT XXXXXXXX XXXXXXXX XXXXXXXX
CARE ------------------------------------------------------------------------------------------------------
COSTS OUTPATIENT XXXXXXXX XXXXXXXX XXXXXXXX
------------------------------------------------------------------------------------------------------------------
ALTERATIONS AND
RENOVATIONS XXXXXXXX XXXXXXXX XXXXXXXX
------------------------------------------------------------------------------------------------------------------
OTHER EXPENSES XXXXXXXX XXXXXXXX XXXXXXXX
------------------------------------------------------------------------------------------------------------------
SUBTOTAL DIRECT COSTS XXXXXXXX XXXXXXXX XXXXXXXX
------------------------------------------------------------------------------------------------------------------
CONSORTIUM/
CONTRACTUAL COSTS XXXXXXXX XXXXXXXX XXXXXXXX
------------------------------------------------------------------------------------------------------------------
TOTAL DIRECT COSTS XXXXXXXX XXXXXXXX XXXXXXXX]
------------------------------------------------------------------------------------------------------------------
TOTAL DIRECT COSTS FOR ENTIRE PROPOSED PROJECT PERIOD (Item 8a)- [XXXXXXXX]
------------------------------------------------------------------------------------------------------------------
JUSTIFICATION (Use continuation pages if necessary):

From Budget for Initial Period: Describe the specific functions of the
personnel, collaborators, and consultants and identify individuals with
appointments that are less than full time for a specific period of the year,
including VA appointments.

For All Years: Explain and justify purchase of major equipment, unusual supplies
requests, patient care costs, alterations and renovations, tuition remission,
and donor/volunteer costs.

From Budget for Entire Period: Identify with an asterisk (*) on this page and
justify any significant increase or decrease in any category over the initial
budget period. Describe any change in effort of personnel.

For Competing Continuation Applications: Justify any significant increases or
decreases in any category over the current level of support.

See Attached Budget Justifications Pages

[Confidential Treatment Requested]

Principal Investigator/Program Director (Last, first, middle):
Went, Xxxxxxx X.
----------------
--------------------------------------------------------------------------------

BUDGET JUSTIFICATIONS - CuraGen Corporation

PERSONNEL

Management
----------
Xx. Xxxxxxx X. Went is the principal investigator and will be responsible for
overall project management, scientific direction, and coordination among
collaborating sites. Dr. Went is a cofounder of CuraGen with Xx. Xxxxxxxx
Xxxxxxxx and has been responsible for research and corporate development. He
currently manages a staff of 24 internally at CuraGen (16 Ph.Ds), as well as 3
subcontracts with leading University researchers. He oversaw the development of
CuraGen's DNA analysis device employed herein. He will devote 20% of his time to
this project.

Xx. Xxxxxxxx X. Xxxxxxxx is the CEO of CuraGen. Xx. Xxxxxxxx'x Ph.D. is in
Molecular Biology and he is the Chief Scientific Officer responsible for
CuraGen's molecular biology efforts. He will devote 10% of his time to the
project to insure that novel molecular methods are integrated into the genome
sequencing efforts at MIT. Xx. Xxxxxxxx will also oversee all contractual
matters related to this collaboration.

Other personnel will be deployed as described below.

Electrophoresis System
----------------------
Xx. Xxxxxxx X. Xxxxxxx will lead the prototyping and development of channel
structures and coordinate activities in Xx. Xxxxxx Xxxxxxxxx'x group. Xx.
Xxxxxxx is a graduate of Berkeley (Xxxx and Soane groups) and is an expert in
macro aspects of micromachined components. He spent 1 year working at Soane
Technologies during his thesis and that experience will aid project coordination
with Soane BioSciences. Xx. Xxxxxxx is experienced both in replication
technologies and MEMs. Under Xx. Xxxxxxx'x direction will be Xx. Xxxxx X.
Xxxxxxx who is responsible for all engineering/machining of electrophoresis
components and Xx. Xxxx X. Xxxxxxx, who is the originator of CuraGen optical,
electrophoresis and base calling technology. A fabrication engineer working
directly with Xx. Xxxxxxx at contract fabrication centers (NNF) will be hired.

Methods development
-------------------
Xx. Xxxxxxx X. XxXxxxx is a molecular biologist carrying out the development of
novel fluorescent protocols for DNA sequencing. Xx. XxXxxxx is experienced in
molecular methods, fluorescence and instrument machining. In addition, Xx.
XxXxxxx is well versed in fluorescent dye strategies, coupling chemistries, and
protocol development. Xx. XxXxxxx will be assisted by a full time technician,
Xxxxxx X. Xxxxxxxxxx. Xx. XxXxxxx will act as a visiting scientist at MIT to
insure close collaboration between methods development and sample preparation.

Informatics
-----------
Xx. Xxxxxx X. Xxxxxxx and Xx. Xxxxxxxxxx Xxxxxxxxxxxxx will lead the refinement
and integration of CuraGen's base calling software into genomic assembly
routines. Xx. Xxxxxxx was the lead programmer on the MacVector product from
Kodak and has extensive experience with commercial software development and
maintenance. Xx. Xxxxxxxxxxxxx who is an expert in pattern analysis will
coordinate the algorithm development and evaluation. Xx. Xxxxxxx'x and Xx.
Xxxxxxxxxxxxx'x efforts reduce to 25% and 10% in years 2 and 3. They will be
assisted by a BS/MS computer scientist.

In Years 2 & 3, the salaries have been increased by 5 percent for inflation.

Fringe benefits have been calculated as 14 percent of salary.

Principal Investigator/Program Director (Last, first, middle):
Went, Xxxxxxx X.
----------------
--------------------------------------------------------------------------------

BUDGET JUSTIFICATIONS - CuraGen Corporation
Page 2

EQUIPMENT

Lab Equipment Electronics for measuring electrical outputs will be required to
test the developing electrophoresis station. An oscilloscope with probes,
capacitance meters, spectrophotometer (for dye QC) are included. Funding remains
available for years 2 and 3.

Power Supplies For electrophoresis experiments, we requests funds for two high
voltage, high power, power supplies. Funding remains available for years
2 and 3.

Software Maintenance of existing graphics and CAD packages. Funding remains
available for years 2 and 3.

Support Hardware Request for hardware for assembly and testing of electrophores
is station. Funding remains available for years 2 and 3.

SUPPLIES

Substrates for fabrication Wafers (Glass and silicon), 500@ $ 55/piece. Expenses
increase 100% in years 2 and 3 as the efforts gear up.

Other fabrication materials/reagents Etchants, coatings. Expenses increase 100%
in years 2 and 3 as the efforts gear up.

Gel Box materials [XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXX]

DNA Sequence supplies [XXXXXXXXXXXXXXXXXXXXXXXX]

Plastic disposables and Test Reagents/chemicals. For general use.

Fluorescent labelling For coupling novel dyes to primers.

TRAVEL

A total of 2 trips to Soane BioSciences @ $1000/each are budgeted for the PI.
Most travel will be to CuraGen, where we have set up a visiting office for Soane
BioSciences. We believe that this level of interaction with our collaborators is
essential. 16 trips (1 week each) are requested to fund travel/expenses
associated with our MIT collaboration. CuraGen has a visiting scientist position
that rotates at MIT.

[Confidential Treatment Requested]

Principal Investigator/Program Director (Last, first, middle):
Went, Xxxxxxx X.
----------------
--------------------------------------------------------------------------------

BUDGET JUSTIFICATIONS - CuraGen Corporation
Page 2

OTHER EXPENSES

Machining/Engineering [XXXXXXXXXXXXXXXXXXX] CuraGen works closely with a number
of design and machining firms in the New Haven area. This number is based on our
historical experience and is adjusted to reflect significant efforts at
delivering a robust and maintenance free system.

Fabrication - NNF [XXXXXXXXXXXXXXXXXXXX] Expense associated with time on
instruments at major fabrication centers, including Cornell's NNF. CuraGen is a
routine user of this facility. Budget increases to 1500 hours in year 2 and
reduces to 1000 hours in year 3.

Publication Costs are estimated at $2,000 per year, as we expect to report on
advances in several technology areas, including separation media, surface design
of plastics for use in DNA electrophoresis, and microreplication methods.

INDIRECT COSTS
Indirect costs are budgeted [XXXXXXX] of direct costs, excluding equipment.

[Confidential Treatment Requested]

DD Principal investigator/Program Director (Last, first, middle):
Went, Xxxxxxx X.
----------------
--------------------------------------------------------------------------------

------------------------------------------------------------------------------------------------------------------------------
DETAILED BUDGET FOR INITIAL BUDGET PERIOD FROM THROUGH
DIRECT COSTS ONLY (Cornell University) 04/01/96 03/31/97
------------------------------------------------------------------------------------------------------------------------------
PERSONNEL (Applicant Organization Only) DOLLAR AMOUNT REQUESTED (omit cents)
------------------------------------------ INST. -----------------------------------------------
ROLE ON TYPE APPT. % EFFORT BASE SALARY FRINGE
NAME PROJECT (months) ON PROJ. SALARY REQUESTED BENEFITS TOTALS
------------------------------------------------------------------------------------------------------------------------------

Principal 12 [XXXX XXXXXX XXXXXX XXXXXX XXXXXXX
Xx. Xxxxxx X. Xxxxxxxxx Investigator 12 XXXX XXXXXX XXXXXX XXXXXX XXXXXXX
------------------------------------------------------------------------------------------------------------------------------
Research
Xx. Xxxxxx Xxxxxx Associate 12 XXXX XXXXXX XXXXXX XXXXXX XXXXXXX
------------------------------------------------------------------------------------------------------------------------------
Research 9 XXXX XXXXXX XXXXXX XXXXXX XXXXXXX
Graduate Student Assistant 3 XXXX XXXXXX XXXXXX XXXXXX XXXXXXX]
------------------------------------------------------------------------------------------------------------------------------

------------------------------------------------------------------------------------------------------------------------------

-------------------------------------------------------------------------------===============================================
SUBTOTALS----------------------------------------- [XXXXXXXX XXXXXXXXX XXXXXXXXX]
-------------------------------------------------------------------------------===============================================

CONSULTANT COSTS
[XXXXXXX]
------------------------------------------------------------------------------------------------------------------------------
EQUIPMENT (Itemize)
Micropositioning Equip; Photomultiplier tubes, housing and power supply; AR Laser;
Filters & gratings; Optical table; Custom optics arrays [XXXXXXX]
------------------------------------------------------------------------------------------------------------------------------
SUPPLIES (Itemize by category)
Substrates, deposition materials, stockroom, machine shop, chemicals and
misc optical components [XXXXXXX]
------------------------------------------------------------------------------------------------------------------------------
TRAVEL

See Budget Justifications Pages [XXXXXXX]
------------------------------------------------------------------------------------------------------------------------------
PATIENT CARE COSTS INPATIENT [XXXXXXX]
-----------------------------------------------------------------------------------------------------
OUTPATIENT [XXXXXXX]
------------------------------------------------------------------------------------------------------------------------------
ALTERATIONS AND RENOVATIONS (Itemize by category)
[XXXXXXX]
------------------------------------------------------------------------------------------------------------------------------
OTHER EXPENSES (Itemize by category)
Publications [XXXXXXX]
Communications (Xerox, Fax, Postage, Phone Tolls) [XXXXXXX]
NNF Charges (Photolithography, Electron Beam Lithography, Etching, Other Fab) [XXXXXXX]
MSC Charges (AFM, SEM, Computer, Thin Film Deposition, etc.) [XXXXXXX] [XXXXXXX]
------------------------------------------------------------------------------------------------------------------------------
SUBTOTAL DIRECT COSTS FOR INITIAL BUDGET PERIOD [XXXXXXX]
---------------------------------------------------------------------------------------------------------------===============
CONSORTIUM/CONTRACTUAL COSTS
DIRECT COSTS TOTAL ------------ [XXXXXXX]
-----------------------------------------------------------
INDIRECT COSTS [XXXXXXX] [XXXXXXX]
---------------------------------------------------------------------------------------------------------------===============
TOTAL DIRECT COSTS FOR INITIAL BUDGET PERIOD (Item 7a, Face Page) ------------ [XXXXXXX]
==============================================================================================================================

[Confidential Treatment Requested]

EE Principal Investigator/Program Director (Last, first, middle):
Went, Xxxxxxx X.
----------------
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------------
BUDGET FOR ENTIRE PROPOSED PROJECT PERIOD
DIRECT COSTS ONLY (Cornell University)
--------------------------------------------------------------------------------------------------------
INITIAL BUDGET ADDITIONAL YEARS OF SUPPORT REQUESTED
BUDGET CATEGORY PERIOD -----------------------------------------------------------
TOTALS (from page 4) 2nd 3rd 4th 5th
--------------------------------------------------------------------------------------------------------

PERSONNEL:
Salary & fringe benefits
Applicant organization only [XXXXXXXX XXXXXXXXX XXXXXXXXX
--------------------------------------------------------------------------------------------------------
CONSULTANT COSTS XXXXXXXX XXXXXXXXX XXXXXXXXX
--------------------------------------------------------------------------------------------------------
EQUIPMENT XXXXXXXX XXXXXXXXX XXXXXXXXX
--------------------------------------------------------------------------------------------------------
SUPPLIES XXXXXXXX XXXXXXXXX XXXXXXXXX
--------------------------------------------------------------------------------------------------------
TRAVEL XXXXXXXX XXXXXXXXX XXXXXXXXX
--------------------------------------------------------------------------------------------------------
PATIENT INPATIENT XXXXXXXX XXXXXXXXX XXXXXXXXX
CARE -------------------------------------------------------------------------------------------
COSTS OUTPATIENT XXXXXXXX XXXXXXXXX XXXXXXXXX
--------------------------------------------------------------------------------------------------------
ALTERATIONS AND
RENOVATIONS XXXXXXXX XXXXXXXXX XXXXXXXXX
--------------------------------------------------------------------------------------------------------
OTHER EXPENSES XXXXXXXX XXXXXXXXX XXXXXXXXX
--------------------------------------------------------------------------------------------------------
SUBTOTAL DIRECT COSTS XXXXXXXX XXXXXXXXX XXXXXXXXX
--------------------------------------------------------------------------------------------------------
CONSORTIUM/
CONTRACTUAL COSTS XXXXXXXX XXXXXXXXX XXXXXXXXX
--------------------------------------------------------------------------------------------------------
TOTAL DIRECT COSTS XXXXXXXX XXXXXXXXX XXXXXXXXX]
------------------------------------------------------------------------------------------==============

TOTAL DIRECT COSTS FOR ENTIRE PROPOSED PROJECT PERIOD (Item 8a)- [XXXXXXX]

------------------------------------------------------------------------------------------==============

JUSTIFICATION (Use continuation pages if necessary):

From budget for Initial Period: Describe the specific functions of the
personnel, collaborators, and consultants and identify individuals with
appointments that are less than full time for a specific period of the year,
including VA appointments.

For All Years: Explain and justify purchase of major equipment, unusual
supplies requests, patient care costs, alterations and renovations, tuition
remission, and donor/volunteer costs.

For Competing Continuation Applications: Justify any significant increases or
decreases in any category over the current level of support.

See Attached Budget Justifications Pages

[Confidential Treatment Requested]

Principal Investigator/Program Director (Last, first, middle):
Went, Xxxxxxx X.
----------------
--------------------------------------------------------------------------------

BUDGET JUSTIFICATIONS - Cornell University

PERSONNEL

The personnel working on this aspect of the project will be Xxxxxxxxx Xxxxxx
Xxxxxxxxx who will oversee and manage the effort at Cornell. He will be
responsible for overall system design and establishing testing paradigms. Xx.
Xxxxxx Xxxxxx is a research associate currently at Cornell who has worked with
the group of Xxxxxxxxx Xxxxxxxxx on the fabrication and optical [XXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]

In Years 2 & 3, the faculty and research associate salaries have been increased
by 4 percent. Graduate student tuition is determined by the University and
increases at a rate of approximately 4%. In academic year 1997-1998, Cornell
will be restructuring its tuition charges which will result in a substantial
reduction in charges for graduate students.

EQUIPMENT

The capital equipment in Year 1 will be used to construct and test the prototype
optical system. [XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXX]

[Confidential Treatment Requested]

Principal Investigator/Program Director (Last, first, middle):
Went, Xxxxxxx X.
----------------
--------------------------------------------------------------------------------

BUDGET JUSTIFICATIONS - Cornell University
Page 2

In Year 2 we will look at optimizing the signal to noise and therefore
throughput of the system and require equipment for analyzing multiple channels
of optical data. Additional optical array components will be required.

[XXXXXXXXXXXXXXXXXXXXXXXXXXXXX XXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXX XXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXX XXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXX XXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXX XXXXXXXX

XXXXXXXXXXXXXXXXXXXXXXXXXXXXXX XXXXXXXX]

In the third year we will be fabricating and testing in-house made components.

SUPPLIES

Substrates, deposition materials, chemicals and misc. optical components. A
major part of this is basic optical components for testing of optical substrates
for our fabricated and custom coated components. In Years 2 & 3, the supplies
costs have been increased by 4 percent for inflation.

TRAVEL

Travel is for three person-trips to scientific conferences and two trips to
CuraGen Corporation each year. In Years 2 & 3, the travel costs have been
increased by 5 percent for inflation.

OTHER EXPENSES

These include local shop, stockroom and lab fees associated with micro-optics
fabrication and testing as well as communication and publication costs
associated with graduate research. In Years 2 & 3, the expenses have been
increased by 4 percent for inflation.

[Confidential Treatment Requested]

DD Principal Investigator/Program Director (Last, first, middle)
Went, Xxxxxxx X.
----------------
--------------------------------------------------------------------------------

DETAILED BUDGET FOR INITIAL BUDGET PERIOD FROM THROUGH
DIRECT COSTS ONLY (Soane BioSciences, Inc.) 04/01/96 03/31/97
-------------------------------------------------------------------------------------------------------------------
PERSONNEL (Applicant Organization Only) % DOLLAR AMOUNT REQUESTED (omit cents)
----------------------------------------- TYPE EFFORT INST. ----------------------------------------------
ROLE ON APPT. ON BASE SALARY FRINGE
NAME PROJECT (months) PROJ. SALARY REQUESTED BENEFITS TOTALS
-------------------------------------------------------------------------------------------------------------------

Principal
Xxxxxxx X. Xxxxxx Investigator 12 [XXX XXXXXXX XXXXXXXXX XXXXXXXXX XXXXXXXXX
-------------------------------------------------------------------------------------------------------------------
Xxxxx X. Xxxxx Scientist 12 XXX XXXXXXX XXXXXXXXX XXXXXXXXX XXXXXXXXX
-------------------------------------------------------------------------------------------------------------------
Xxxxxxx Xxxxxx Amigo Scientist 12 XXX XXXXXXX XXXXXXXXX XXXXXXXXX XXXXXXXXX
-------------------------------------------------------------------------------------------------------------------
Molecular
Xxxxxxxxx X. Xxxxx Biologist 12 XXX XXXXXXX XXXXXXXXX XXXXXXXXX XXXXXXXXX
-------------------------------------------------------------------------------------------------------------------
Xxxxxx Xxxx Scientist 12 XXX XXXXXXX XXXXXXXXX XXXXXXXXX XXXXXXXXX
-------------------------------------------------------------------------------------------------------------------
Polymer
Xxx Xxx Chemist 12 XXX XXXXXXX XXXXXXXXX XXXXXXXXX XXXXXXXXX
-------------------------------------------------------------------------------------------------------------------
Designer
Xxxxx Xxxxxxxxx Machinist 12 XXX XXXXXXX XXXXXXXXX XXXXXXXXX XXXXXXXXX
-------------------------------------------------------------------------------------------------------------------
Polymer
To be determined Engineer 12 XXX XXXXXXX XXXXXXXXX XXXXXXXXX XXXXXXXXX]
--------------------------------------------------------------------===============================================
SUBTOTALS----------------------------- [XXXXXXXXX] [XXXXXXXXX] [XXXXXXXXX]
--------------------------------------------------------------------===============================================

CONSULTANT COSTS
[XXXXXXXXX]
-------------------------------------------------------------------------------------------------------------------
EQUIPMENT (Itemize)
Thin Slab Sequencer [XXXXXXXX]
UV/VIS Spectrophotometer [XXXXXXXX]
Flouorescent Spectrophotometer [XXXXXXXX]
Fabrication of curing &
spin-coating stations [XXXXXXXX] [XXXXXXX]
-------------------------------------------------------------------------------------------------------------------
SUPPLIES (Itemize by category)
Sequencing Kits, Template, Dyes [XXXXXX]
Monomers, Polymers, Solvents, Buffers [XXXXXX]
Miscellaneous Chemicals & Supplies [XXXXXX] [XXXXXX]
-------------------------------------------------------------------------------------------------------------------
TRAVEL
Travel to CuraGen Corporation and Key Conferences [XXXXXXX]
-------------------------------------------------------------------------------------------------------------------
PATIENT CARE COSTS INPATIENT [XXXXXXX]
----------------------------------------------------------------------------------------
OUTPATIENT [XXXXXXX]
-------------------------------------------------------------------------------------------------------------------
ALTERATIONS AND RENOVATIONS (Itemize by category)
[XXXXXXX]
-------------------------------------------------------------------------------------------------------------------
OTHER EXPENSES (Itemize by category)
Publication Costs [XXXXXXX]
-------------------------------------------------------------------------------------------------------------------
SUBTOTAL DIRECT COSTS FOR INITIAL BUDGET PERIOD [XXXXXXX]
-------------------------------------------------------------------------------------------------------============
CONSORTIUM/CONTRACTUAL COSTS
DIRECT COSTS TOTAL---- [XXXXXXX]
-----------------------------------------------------
INDIRECT COSTS [XXXXXXXXXX XXXXXXXXXXX]
-------------------------------------------------------------------------------------------------------============
TOTAL DIRECT COSTS FOR INITIAL BUDGET PERIOD (Item 7a, Face Page)---- [XXXXXX]
-------------------------------------------------------------------------------------------------------============

[Confidential Treatment Requested]

EE Principal Investigator/Program Director (Last, first, middle)
Went, Xxxxxxx X.
----------------

-----------------------------------------------------------------------------------------------------------
BUDGET FOR ENTIRE PROPOSED PROJECT PERIOD
DIRECT COSTS ONLY (Soane BioSciences, Inc.)

-----------------------------------------------------------------------------------------------------------
BUDGET CATEGORY INITIAL BUDGET PERIOD ADDITIONAL YEARS OF SUPPORT REQUESTED
----------------------------------------------------------
TOTALS (from page 4) 2nd 3rd 4th 5th
-----------------------------------------------------------------------------------------------------------

PERSONNEL:
Salary & fringe benefits
Applicant organization only [XXXXXXXX XXXXXXXXX XXXXXXXX
-----------------------------------------------------------------------------------------------------------
CONSULTANT COSTS XXXXXXXX XXXXXXXXX XXXXXXXX
-----------------------------------------------------------------------------------------------------------
EQUIPMENT XXXXXXXX XXXXXXXXX XXXXXXXX
-----------------------------------------------------------------------------------------------------------
SUPPLIES XXXXXXXX XXXXXXXXX XXXXXXXX
-----------------------------------------------------------------------------------------------------------
TRAVEL XXXXXXXX XXXXXXXXX XXXXXXXX
-----------------------------------------------------------------------------------------------------------
PATIENT INPATIENT XXXXXXXX XXXXXXXXX XXXXXXXX
CARE ----------------------------------------------------------------------------------------------
COSTS OUTPATIENT XXXXXXXX XXXXXXXXX XXXXXXXX
-----------------------------------------------------------------------------------------------------------
ALTERATIONS AND
RENOVATIONS XXXXXXXX XXXXXXXXX XXXXXXXX
-----------------------------------------------------------------------------------------------------------
OTHER EXPENSES XXXXXXXX XXXXXXXXX XXXXXXXX
-----------------------------------------------------------------------------------------------------------
SUBTOTAL DIRECT COSTS XXXXXXXX XXXXXXXXX XXXXXXXX
-----------------------------------------------------------------------------------------------------------
CONSORTIUM/
CONTRACTUAL COSTS XXXXXXXX XXXXXXXXX XXXXXXXX
-----------------------------------------------------------------------------------------------------------
TOTAL DIRECT COSTS XXXXXXXX XXXXXXXXX XXXXXXXX]
-----------------------------------------------------------------------------------------------------------
TOTAL DIRECT COSTS FOR ENTIRE PROPOSED PROJECT PERIOD (Item 8a)- [XXXXXXXXXX]
-----------------------------------------------------------------------------------------------------------
JUSTIFICATION (Use continuation pages if necessary):

For All Years: Explain and justify purchase of major equipment, unusual supplies
requests, patient care costs, alterations and renovations, tuition remission,
and donor/volunteer costs.

For Competing Continuation Applications: Justify any significant increases or
decreases in any category over the current level of support.

See Attached Budget Justifications Pages

[Confidential Treatment Requested]

Principal Investigator/Program Director (Last, first, middle):
Went, Xxxxxxx X.
----------------
--------------------------------------------------------------------------------

BUDGET JUSTIFICATIONS - Soane BioSciences, Inc.

PERSONNEL

Xx. Xxxxxx will be responsible for project management at Soane BioSciences,
Inc., as well as scientific direction, and coordination with the collaborating
sites. Xx. Xxxxx will provide scientific guidance in the areas of
electrophoresis media, polymer formulation, replication and microfabrication.
Other personnel will be deployed as described below.

Media development
-----------------
Xx. Xxxx Xxxxx will lead the media development effort. He is an expert in
hydrogels, including temperature-responsive gels, with considerable
electrophoresis experience. He will be responsible for overall experimental
design, and will directly conduct experiments on gel phase behavior, stability,
and polymerization of media in the microreplicated systems. Xxxxx Xxxx will
prepare DNA samples and perform sequencing runs to evaluate gel performance,
activities with which he has significant experience. Xx. Xxxxx Xxx will be
responsible for monomer synthesis and for synthesizing novel polymer structures,
such as the graft polymers required for the dynamic porosity gels. Xx. Xxx is an
expert in monomer and polymer synthesis. It is expected that Xx. Xxx will spend
about 50% of his time on media development, and the other half of his time on
Microreplication development.

Microreplication development
----------------------------
Xx. Xxxxxxx Xxxxxx will lead the Microreplication effort at SBio, which will
consist of developing base-substrate bonding methods, substrate prototyping via
replication, polymer formulation, and channel surface design and testing. Xx.
Xxxxxx is an accomplished polymer scientist with extensive experience in
polymerization methods, formulation, surface modification and capillary
electrophoresis. Xx. Xxxxxx also has extensive experience in hydrogels from
previous research on contact lenses, and she will consult on the gel development
efforts. Xx. Xxxxx Xxx will spend 50% time on this project, assisting Xx. Xxxxxx
in the formulation and surface modification efforts. A polymer engineer will be
hired for design and construction of UV curing and spin coating stations, along
with testing of replication processes. Xxxxx Xxxxxxxxx, an experienced
mechanical designer and machinist, will assist the polymer engineer during
design and construction of curing and spin-coating stations, performing
requisite machining tasks. Xx. Xxxxxxxxx will also machine plastic plates for
testing in the thin slab system, and perform miscellaneous design/machining
tasks.

In Years 2 & 3, the salaries have been increased by 4 percent for inflation.

Fringe benefits have been calculated as 14 percent of salary.

EQUIPMENT

A prototype of CuraGen's thin slab sequencer is budgeted at [XXXXXX], which will
enable efficient evaluation of media separation properties and testing of
plastic substrates. A UV/VIS (Xxxxxx Xxxxx model Lambda 11 or equivalent) and a
fluorescence spectrophotometer (PE LX50B or equivalent) are budgeted for
characterizing the optical properties of plastic substrate formulations. We have
budgeted the purchase of components for constructing UV curing and spin-coating
stations, including UV lamp, power supply, motor and mechanical parts. The UV
curing station will be used for replication experiments, and for UV
polymerization of media within the microreplicated systems.

[Confidential Treatment Requested]

Principal Investigator/Program Director (Last, first middle):
Went, Xxxxxxx X.
----------------
--------------------------------------------------------------------------------

BUDGET JUSTIFICATIONS - Soane BioSciences, Inc.
Page 2

SUPPLIES

Sequencing reagents will be needed to evaluate gel performance, and [XXXXXXXXXX
XXXXXXXXXXXXXXXXX] We budgeted [XXXXXXX] for the purchase of monomers, polymers,
solvents and buffers required in both gel development and microreplication
development. Miscellaneous supplies include machine tools, contract analytical
services (e.g., for specialized surface characterization), replacement parts or
necessary accessories for equipment used in this project, etc.

TRAVEL

A total of 7 trips to CuraGen [XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXX]

OTHER EXPENSES

Publication costs are estimated at $2,000 per year, as we expect to report on
advances in several technology areas, including separation media, surface design
of plastics for use in DNA electrophoresis, and microreplication methods.

INDIRECT COSTS

[XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]

[Confidential Treatment Requested]

DD Principal Investigator/Program Director (Last, first, middle)
Went, Xxxxxxx, T.
-----------------
--------------------------------------------------------------------------------

---------------------------------------------------------------------------------------------------------------------------
DETAILED BUDGET FOR INITIAL BUDGET PERIOD FROM THROUGH
DIRECT COSTS ONLY (The Xxxxxxxxx Institute) 04/01/96 03/31/97
---------------------------------------------------------------------------------------------------------------------------
PERSONNEL (Applicant Organization Only) DOLLAR AMOUNT REQUESTED (omit cents)
--------------------------------------- -----------------------------------------
TYPE
ROLE ON APPT. % EFFORT INST BASE SALARY FRINGE
NAME PROJECT (months) ON PROJ. SALARY REQUESTED BENEFITS TOTALS
---------------------------------------------------------------------------------------------------------------------------

Xxxxxx X. Xxxxxxx Principal Investigator 12 [XX] [XXXXXX] [XXXXXX] [XXXXXXX] [XXXXXXX]
---------------------------------------------------------------------------------------------------------------------------
Lincoln X. Xxxxx Research Scientist 12 [XX] [XXXXXX] [XXXXXX] [XXXXXXX] [XXXXXXX]
---------------------------------------------------------------------------------------------------------------------------
Xxxxx Xxxx Technical Assistant 12 [XX] [XXXXXX] [XXXXXX] [XXXXXXX] [XXXXXXX]
---------------------------------------------------------------------------------------------------------------------------
Xxxxxxx Xxx Research Specialist 12 [XX] [XXXXXX] [XXXXXX] [XXXXXXX] [XXXXXXX]
---------------------------------------------------------------------------------------------------------------------------

---------------------------------------------------------------------------------------------------------------------------

SUBTOTALS---------[XXXXXXX] [XXXXXXXX] [XXXXXXXXX]
---------------------------------------------------------------------------------------------------------------------------

CONSULTANT COSTS
[XXXXX]
-------------------------------------------------------------------------------
EQUIPMENT (Itemize)
Prototype 9mm spacing coated pins for solid-phase additions
(3 @ $5,000/each)) [XXXXXX]

SUPPLIES (Itemize by category)
Magnetic particles, custom synthesis [XXXXXX]
Sequencing reagents (5,000 @ $1/each) [XXXXXX]
Magnets, donuts, dots, bars, custom [XXXXXX]
Plastic plates, tips, etc. [XXXXXX] [XXXXXX]
--------------------------------------------------------------------------------
TRAVEL

[XXXXXX]
--------------------------------------------------------------------------------
PATIENT CARE COSTS INPATIENT [XXXXXX]
-------------------------------------------------------
OUTPATIENT [XXXXXX]
--------------------------------------------------------------------------------
ALTERATIONS AND RENOVATIONS (Itemize by category)
[XXXXXX]
--------------------------------------------------------------------------------
OTHER EXPENSES (Itemize by category)
Phone $1,000
Photocopying $200
Mail $500
Publication costs $500 [XXXXXX]
--------------------------------------------------------------------------------
SUBTOTAL DIRECT COSTS FOR INITIAL BUDGET PERIOD [XXXXXX]
--------------------------------------------------------------------------------
CONSORTIUM/CONTRACTUAL COSTS
DIRECT COSTS TOTAL---- [XXXXXX]
-----------------------------------------------
INDIRECT COSTS [XXXXXXXXXXXXXXXXX]
--------------------------------------------------------------------------------

TOTAL DIRECT COSTS FOR INITIAL BUDGET PERIOD (Item 7a, Face Page)---- [XXXXXX]
--------------------------------------------------------------------------------

[Confidential Treatment Requested]

EE Principal Investigator/Program Director (Last, first, middle)
Went, Xxxxxxx X.
----------------
--------------------------------------------------------------------------------
BUDGET FOR ENTIRE PROPOSED PROJECT PERIOD
DIRECT COSTS ONLY (The Xxxxxxxxx Institute)

--------------------------------------------------------------------------------

BUDGET CATEGORY INITIAL BUDGET ADDITIONAL YEARS OF SUPPORT REQUESTED
PERIOD ------------------------------------------------------------
TOTALS (from page 4) 2nd 3rd 4th 5th
---------------------------------------------------------------------------------------------------------

PERSONNEL:
Salary & fringe benefits
Applicant organization only [XXXXXX] [XXXXXX] [XXXXXX]
---------------------------------------------------------------------------------------------------------
CONSULTANT COSTS [XXXXXX] [XXXXXX] [XXXXXX]
---------------------------------------------------------------------------------------------------------
EQUIPMENT [XXXXXX] [XXXXXX] [XXXXXX]
---------------------------------------------------------------------------------------------------------
SUPPLIES [XXXXXX] [XXXXXX] [XXXXXX]
---------------------------------------------------------------------------------------------------------
TRAVEL [XXXXXX] [XXXXXX] [XXXXXX]
---------------------------------------------------------------------------------------------------------
PATIENT INPATIENT [XXXXXX] [XXXXXX] [XXXXXX]
CARE -------------------------------------------------------------------------------------------
COSTS OUTPATIENT [XXXXXX] [XXXXXX] [XXXXXX]
---------------------------------------------------------------------------------------------------------
ALTERATIONS AND
RENOVATIONS [XXXXXX] [XXXXXX] [XXXXXX]
---------------------------------------------------------------------------------------------------------
OTHER EXPENSES [XXXXXX] [XXXXXX] [XXXXXX]
---------------------------------------------------------------------------------------------------------
SUBTOTAL DIRECT COSTS [XXXXXX] [XXXXXX] [XXXXXX]
---------------------------------------------------------------------------------------------------------
CONSORTIUM/
CONTRACTUAL COSTS [XXXXXX] [XXXXXX] [XXXXXX]
---------------------------------------------------------------------------------------------------------
TOTAL DIRECT COSTS [XXXXXX] [XXXXXX] [XXXXXX]
-------------------------------------------------------------------------------------------==============
TOTAL DIRECT COSTS FOR ENTIRE PROPOSED PROJECT PERIOD (Item 8a)-- [XXXXXXX]
-------------------------------------------------------------------------------------------==============

JUSTIFICATION (Use continuation pages if necessary):

For All Years: Explain and justify purchase of major equipment, unusual
supplies requests, patient care costs, alterations and renovations, tuition
remission, and donor/volunteer costs.

For Competing Continuation Applications: Justify any significant increases or
decreases in any category over the current level of support.

See Attached Budget Justifications Pages

[Confidential Treatment Requested]

Principal Investigator/Program Director (Last, first, middle):
Went, Xxxxxxx X.
---------------
-------------------------------------------------------------------------------

BUDGET JUSTIFICATIONS - The Xxxxxxxxx Institute

PERSONNEL

Year 1
Xxxxxx X. Xxxxxxx (Research Scientist and group head of the Center's DNA
sequencing and automation group) [XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXX]

Xxxxxxx Xxxxx (Research Scientist) is Director of Informatics at the Xxxxxxxxx
Institute/MIT Center for Genome Research. [XXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXX]

Xxxxx Xxxx (Technical Assistant) will be committed to the project [XXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]

Xxxxxxx Xxx (Research Specialist) will be committed to this project [XXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]

Year 2
As above, with the exception of the reduction of Xxxxx Xxxx and Xxxxxxx Xxx to
[XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXX]

Year 3
As Year 2. In Year 3, the salaries have been increased by 4 percent for
inflation.

[XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]

[Confidential Treatment Requested]

BUDGET JUSTIFICATIONS - The Xxxxxxxxx Institute
Page 2

EQUIPMENT

Year 1
Qty Item Unit Price Total Price

3 Prototype 9mm spacing coated pins for
solid-phase additions $5,000 $15,000

Our plan for the solid-phase loading is to use a reusable 12 channel
9mm spacing pin set. This allows us to move from any standard 96, 192 or 384
well plate to the gel electrophoresis system without modification. We expect to
make three basic types of pins, one for SPRI, one for Biotin-streptavidin and
one for digoxigenin-anti-digoxigenin.

Year 2

Qty Item Unit Price Total Price
1 9mm spacing pins for solid or liquid-phase
additions $10,000 $10,000

The exact design of the 9mm tool head will be the subject of our work
in year 1. At this point we would redesign and improve our best approach from
year 1 to enable us to use the system in year 2. The increased cost of this tool
head reflects the fact that in year 1 we built prototype units.

We will use existing robotic systems for development, to show our strong
commitment to the project.

SUPPLIES

In Years 2 & 3, the supplies costs have been increased by 4 percent for
inflation.

OTHER EXPENSES

In Years 2 & 3, the other expenses will be adjusted 4 percent for inflation.

FF Principal Investigator/Program Director (Last, first, middle):
Went, Xxxxxxx X.
----------------
--------------------------------------------------------------------------------
BIOGRAPHICAL SKETCH
Give the following information for the key personnel and consultants and
collaborators. Begin with the principal investigator/program director.
Photocopy this page for each person.

--------------------------------------------------------------------------------
NAME POSITION TITLE
Xxxxxxx X. Went Vice President

YEAR
INSTITUTION AND LOCATION DEGREE CONFERRED FIELD OF STUDY
-------------------------------------------------------------------------------------------------------------

Xxxxxxxx-Xxxxxx University, Pittsburgh, PA B.S. 1985 Chemical Engineering
University of California, Berkeley, Berkeley, CA Ph.D. 1991 Chemical Engineering
Cornell University, Ithaca, NY PostDoc 1993 Chemical Physics
-------------------------------------------------------------------------------------------------------------

RESEARCH AND/OR PROFESSIONAL EXPERIENCE: Concluding with present position, list
in chronological order previous employment, experience, and honors. Key
personnel include the principal investigator and any other individuals who
participate in the scientific development or execution of the project. Key
personnel typically will include all individuals with doctoral or other
professional degrees, but in some projects will include individuals at the
masters or baccalaureate level provided they contribute in a substantive way to
the scientific development or execution of the project. Include present
membership on any Federal Government public advisory committee. List, in
chronological order, the titles, all authors, and complete references to all
publications during the past three years and to representative earlier
publications pertinent to this application. DO NOT EXCEED TWO PAGES.

Professional Experience:
-----------------------

1993-1994 Director of Structural Biology
CuraGen Corporation, Branford, CT

1994-Present Vice President
CuraGen Corporation, Branford, CT

Honors and Awards:
-----------------

Xxxx Summer Research Program, 1984
University Honors, 1985

Bibliography:
------------

1. "Microfabricated Device for High-Resolution Multiplex DNA Analysis",
G.T.Went, and X.X.Xxxxxxx, to be submitted.

2. "Imaging Spectrograph for DNA Analysis",X.X. Xxxxxxx, and G.T. Went,
Analytical Chemistry (1995), to be submitted.

3. "Algorithm for DNA Sequence Determination", X.X.Xxxxxxx, X.X. Xxxxxx, and
G.T. Went, in preparation (1995).

4. "Standard Absorption Isotherms for Nitrogen and Methane on Graphite:
Improved Resolution at Low Pressure", X.X. Xxxxxxx, G.T. Went, X.X. Xxxxxx
and X.X. Xxxxxxx, submitted for publication, Langmuir (June 1995)

5. "Oxidation of Reduced Platinum Clusters in Pt-NaY Catalysts", X.X. Xxxxxxx,
G.T. Went, R.Csenscits, A.T. Xxxx, X.X. Xxxxxxxx, and X.X. Xxxxx, accepted
for publication, Journal of Catalysis (1994).

6. "A Physico-Chemical Study of the Aging of Zeolite Synthesis Gels", X.X.
Xxxxxx, G.T. Went, X.X. Xxxxx, and A.T. Xxxx, Zeolites 12 (1992) 733.

--------------------------------------------------------------------------------

7. "Laser Raman Spectroscopy of NH3 and ND3 adsorbed on TiO2 (Anatase)", G.T.
Went, A.T. Xxxx, Catalysis Letters 11, 111 (1992).

8. "Quantitative Structural Analysis of Dispersed Vanadia Species in TiO2
(Anatase)- Supported V2O5", G.T. Went, L. Leu, X. Xxxxxxxx, A.T. Xxxx,
Journal of Catalysis 134, 479 (1992).

Invited Presentations (16 total):
--------------------------------

"Engineering Novel Pharmaceuticals," Invited Lecture, Department of Chemical
Engineering, University of California, Santa Barbara, CA, May 18, 1995.

"Microfabricated Device for DNA Fragment Analysis," Invited Lecture, BioChips
Conference, Washington DC, May 10, 1995.

"Modular Platform for DNA Fragment Analysis," Invited Lecture, DNA Sequencing,
Mapping and Bioinformatics Conference, San Francisco, CA, July, 1994.

"Kinetic, Thermodynamic and Spectroscopic Studies of Surface Phenomena", Invited
Lecture, Department of Chemical Engineering, Purdue University, Purdue, IL,
November, 1992.

Patents:
-------

"Apparatus and Method for the Generation, Separation, Detection, and Recognition
of Biopolymer Fragments", X.X. Xxxxxxx, X.X. Xxxxxxxx, and G.T. Went, U.S.
and Foreign Patent Pending.

"Consensus Configurational Bias Monte Carlo Method and System for Pharmacophore
Structure Determination", X.X. Xxxx, X.X. Xxxxxxxx, and G.T. Went, U.S. and
Foreign Patent Pending.

Give the following information for the key personnel and consultants and
collaborators. Begin with the principal investigator/program director.
Photocopy this page for each person.

--------------------------------------------------------------------------------
NAME POSITION TITLE
Xxxxxxxx X. Xxxxxxxx Chief Executive Officer

YEAR
INSTITUTION AND LOCATION DEGREE CONFERRED FIELD OF STUDY
------------------------------------------------------------------------------------------------------------

Xxxxxxxx-Xxxxxx University, Pittsburgh, PA B.S. 1985 Chemical Engineering
Yale University, New Haven, CT Ph.D. 1991 Molecular Biology
Yale University, New Haven, CT PostDoc 1993 Molecular Biology
------------------------------------------------------------------------------------------------------------

Professional Experience:
-----------------------

1988-Present Member of the Board of Directors
Laticrete International, Inc., Xxxxxxx, CT

1991-Present Chairman of the Board of Directors
CuraGen Corporation, Branford, CT

1993-Present Chief Executive Officer
CuraGen Corporation, Branford, CT

Honors and Awards:
-----------------

National Research Service Award, NIH, 1985-1989
Xxxx Xxxxxxxx Xxxxxxxx Prize for the Outstanding Doctoral candidate in
Experimental Zoology, 1991

Bibliography:
------------

1. "Modularity of the Slit Protein", X. X. Xxxxxxxx, and S. Artavanis-
Tsakonas, S., J. Mol. Biol. 227, 367-370 (1992).

2. "Gene Patents", X.X. Xxxxxxxx, Nature 356, 738 (1992).

3. "slit: an Extracellular Protein Necessary for the Development of Midline
Glia and Axon Pathways of the Central Nervous System contains both EGF and
Flank-LRR-Flank Domains", X.X. Xxxxxxxx, Ph.D. Thesis, Yale University
(1991).

4. "slit: an Extracellular Protein Necessary for Development of Midline Glia
and Commissural Axon Pathways Contains both EGF and LRR Domains", X.X.
Xxxxxxxx, X.X. Xxxxxx, X.X. Xxxxxxx, and S. Artavanis-Tsakonas, Genes &
Development 4, 2169-2187 (1990).

5. "slit: An EGF-Homologous Locus of D. melanogaster Involved in the
Development of the Embryonic Central Nervous System", X.X. Xxxxxxxx, X. X.
Xxxxxxx, X. Xxxxxxx, and S. Artavanis-Tsakonas, Cell 55, 1047-1059 (1988).

Patents:
-------

"Apparatus and Method for the Generation, Separation, Detection, and Recognition
of Biopolymer Fragments", X.X. Xxxxxxx, X.X. Xxxxxxxx, and G.T. Went, U.S.
and Foreign Patent Pending.

"Consensus Configurational Bias Monte Carlo Method and System for Pharmacophore
Structure Determination", X.X. Xxxx, X.X. Xxxxxxxx, and G.T. Went, U.S. and
Foreign Patent Pending.

"Purified slit Protein and Sequence Elements thereof", X.X. Xxxxxxxx, and S.
Artavanis-Tsakonas, US Patent Pending, (1990).

Give the following information for the key personnel and consultants and
collaborators. Begin with the principal investigator/program director.
Photocopy this page for each person.

--------------------------------------------------------------------------------
NAME POSITION TITLE
Xxxxxxx X. XxXxxxx Senior Research Scientist

YEAR
INSTITUTION AND LOCATION DEGREE CONFERRED FIELD OF STUDY
------------------------------------------------------------------------------------------------

Xxxxxxxx-Xxxxxx University, Pittsburgh, PA B.S. 1984 Molecular Biology
Yale University, New Haven, CT Ph.D. 1993 Biology
------------------------------------------------------------------------------------------------

Professional Experience:
-----------------------

1984-1986 Research Associate
Max-Xxxxxx-Institute-fur-Entwicklungsbiogiogie, Tubingen, Germany

1992-1994 Graduate Scholar
Miles Biotechnology, West Haven, CT

1994-Present Senior Research Scientist
CuraGen Corporation, Branford, CT

Bibliography:
------------
1. "A Single Amino Acid Distinguishes the Interaction of Two Rhinovirus
Stereotypes with Their Receptor, ICAM-1," X.X. XxXxxxx and X. Xxxxx, in
preparation (1994).

2. "Putative Drosophila pheromone-Binding Proteins Expressed in a Subregion of
the Olfactory System," X.X. XxXxxxx, D.S. Heknat-Xxxxx, X. Xxxxxx, and X.
Xxxxxxx, J. Biol. Chem. in press, (1994).

3. "A Simple Chemosensory Response in Drosophila and the Isolation of Acj
Mutants in Which it is Affected," X.X. XxXxxxx, M.P. Monte, X.X. Xxxxxxx,
X. Xxxxxxx, and X. Xxxxxxx, Proc. Nat. Acad. Sci. 86, 8118-8122 (1989).

4. "Growth Cone Behavior on Gradients of Substratum Bound Laminin," X.X.
XxXxxxx, and X. X. Xxxxx, Dev. Biology 130, 232-236 (1988).

5. "A Transient Rise in Cytosolic Calcium Follows Stimulation of Quiescent
Cell with Growth Factors and is Inhabitable with Phorbal Myristate
Acetate," X.X. XxXxxx, X.X. XxXxxxx, and X. X. Xxxxxx, J. Cell Biology 101,
372-379 (1985).

--------------------------------------------------------------------------------

Give the following information for the key personnel and consultants and
collaborators. Begin with the principal investigator/program director.
Photocopy this page for each person.

--------------------------------------------------------------------------------
NAME POSITION TITLE
Xxxxxxx X. Xxxxxxx Senior Research Scientist

YEAR
INSTITUTION AND LOCATION DEGREE CONFERRED FIELD OF STUDY
-------------------------------------------------------------------------------------------------------------

Xxxxxxxx-Xxxxxx University, Pittsburgh, PA B.S. 1989 Chemical Engineering
University of California, Berkeley, Berkeley, CA Ph.D. 1995 Chemical Engineering
-------------------------------------------------------------------------------------------------------------

Professional Experience:
-----------------------
1988 Research Engineer
Air Products and Chemicals, Allentown, PA

1989 Research Assistant
Carnegie Mellon University, Xxxxxxxxxx, XX

0000-0000 Project Engineer
Soane Technologies, Inc., Hayward, CA

1995-Present Senior Research Scientist
CuraGen Corporation, Branford, CT

Honors and Awards:
-----------------

American Institute of Chemical Engineers Chapter Award for Scholastic
Achievement, 1987
Xxxxxx Xxxxxxxx Society Award Finalist, 1989
American Institute of Chemists Foundation Award, 1989

--------------------------------------------------------------------------------

Give the following information for the key personnel and consultants and
collaborators. Begin with the principal investigator/program director.
Photocopy this page for each person.

--------------------------------------------------------------------------------
NAME POSITION TITLE
Xxxxx X. Xxxxxxx Senior Research Engineer

YEAR
INSTITUTION AND LOCATION DEGREE CONFERRED FIELD OF STUDY
-------------------------------------------------------------------------------------------------------------

Xxxxxxxxxx xx Xxxxxxxxxxxx, Xxxxxxxxxxxx, Xxxxxx B.S. 0000 Xxxxxxxxx
Xxxxxxxxxx xx Xxxxxxxxxxxx, Xxxxxxxxxxxx, Xxxxxx X.Xx. 0000 Xxxxxxxxx
Xxxxxxxxxx xx Xxxxxxx, Xxxxxx, XX Ph.D. 0000 Xxxxxxxxx
Xxxxxxx Xxxxxxxxxx, Xxxxxx, XX PostDoc 1995 Chemistry
-------------------------------------------------------------------------------------------------------------

Professional Experience:
-----------------------

1983-1985 Assistant
Global Thermoelectric Generator Systems, Ltd, Alberta, Canada

1995-Present Senior Research Engineer
CuraGen Corporation, Branford, CT

Bibliography:
------------

1. "The Preparation and Crystal Structure of Tetrakis (phenylmethoxy methane",
X.X. Xxxxxxx, X.X. Xxxx, X.X. Xxxxx, and X.X. Xxxxxxxx, Can. J. Chem., 67,
143 (1989).

2. "The ~B - ~X Laser-Induced Fluroescence Excitation Spectrum of Jet-Cooled
Cl2CS: Origin Location and Partial Vibronic Analysis", X. Xxxxxxxxx, X.X.
Xxxxxxx, and X.X. Xxxxx, J. Mol. Spectrosc., 147, 414 (1991).

3. "Ion-molecule Reaction Studies Below 10 K: The State Specific Reactions of
Ar+ (2PJ) with H2, D2 and HD", M. A. Xxxxx, X. Xxxxxx, X.X. Xxxxxxx, and X.
X. Xxxxxx, Acta Physica Universitatis Comenianae, XXXIV 3 (1994).

4. "Direct Observation of HSF6+ and the Bracketing of the SF6 Proton Affinityt
5 K", X. X. Xxxxxxx and M. A. Xxxxx, J. Chem. Phys., 101, 3410 (1994).

5. "Electronic Energy Transfer Kinetics of Xe+ (2P1/2) at Very Low
Temperature", X. X. Xxxxxxx, and M.A.Xxxxx, J. Chem. Phys., 101, 3852
(1994).

6. "Gas-Phase Reactions Relevant for Plasma Deposition of Diamond:
Dissociative Charge Transfer of Ar+ ions with CH4 in the Energy Range From
10-4 from 2 e V", X. Xxxx, X. Xxxxxxxxxxx, X. Xxxxxxxx, X. Xxxxxxxx, X.
Xxxxx, X. X. Xxxxxxx, and M. A. Xxxxx, manuscript in preparation.

--------------------------------------------------------------------------------

Give the following information for the key personnel and consultants and
collaborators. Begin with the principal investigator/program director.
Photocopy this page for each person.

--------------------------------------------------------------------------------
NAME POSITION TITLE
Xxxx X. Xxxxxxx Senior Research Engineer

YEAR
INSTITUTION AND LOCATION DEGREE CONFERRED FIELD OF STUDY
-------------------------------------------------------------------------------------------------------------

California Institute of Technology, Pasadena, CA B.S. 1987 Mathematics
California Polytechnic State University, San Xxxx M.S. 1991 Mechanical Eng.
Obispo, CA
-------------------------------------------------------------------------------------------------------------

Professional Experience:
-----------------------
1988-1989 Assistant in Research and Instruction
Xxxxxxxxx Xxxxxxxxxx, Xxxxxxxxx, XX

0000 Research Associate
California Polytechnic State University, San Luis Obispo, CA

1994-Present Senior Research Engineer
CuraGen Corporation, Branford, CT

Honors and Awards:
-----------------
Caltech Prize Scholarship, 1985
Xxxxxxx X. Xxxxx Memorial Scholarship, 1986
Mobil Fellowship and First Year Merit Prize, 1987

Bibliography:
------------

1. "Microfabricated Device for High-Resolution Multiplex DNA Analysis",
G.T.Went, and X.X.Xxxxxxx, to be submitted.

2. "Imaging Spectrograph for DNA Sequencing", X. X.Xxxxxxx and G. T. Went,
manuscript in preparation, Analytical Chemistry (1995).

3. "Algorithm for DNA Sequence Determination," X.X. Xxxxxxx, X.X. Xxxxxx, and
G.T. Went, in preparation (1995).

Patents:
-------

"Apparatus and Method for the Generation, Separation, Detection, and Recognition
of Biopolymer Fragments", X.X. Xxxxxxx, X.X. Xxxxxxxx, and G.T. Went, U.S.
and Foreign Patent Pending.

Give the following information for the key personnel and consultants and
collaborators. Begin with the principal investigator/program director.
Photocopy this page for each person.

--------------------------------------------------------------------------------
NAME POSITION TITLE
Xxxxxxxxxx Xxxxxxxxxxxxx Director of Bioinformatics

YEAR
INSTITUTION AND LOCATION DEGREE CONFERRED FIELD OF STUDY
--------------------------------------------------------------------------------------------------------------

Belgrade University, Belgrade, Yugoslavia B.S. 1984 Electrical Engineering
Santa Xxxxx University, Santa Clara, CA X.Xx. 1986 Computer Science
University of California, Santa Xxxx, Santa Cruz, CA Ph.D 1990 Computer and
Informational Sciences
--------------------------------------------------------------------------------------------------------------

Professional Experience:
-----------------------

1984-1985 Research Assistant and Lecturer
Xxxxx Xxxxx Xxxxxxxxxx, Xxxxx Xxxxx, XX

0000-0000 Research and Teaching Assistant
University of California, Xxxxx Xxxx, Xxxxx Xxxx, XX

0000-0000 Adjunct Lecturer
Xxxxx Xxxxx Xxxxxxxxxx, Xxxxx Xxxxx, XX

0000-0000 Computer Scientist
Xxxxx Xxxxxxx Institute of Science and Medicine, Xxxx Xxxx, XX

0000-0000 Assistant Scientist
Argonne National Laboratory, Argonne, IL

1995-Present Director of Bioinformatics
CuraGen Corporation, Branford, CT

Bibliography:
------------

1. "Learning in the Presence of Background Knowledge", X. Xxxxxxxxxxxxx,
Technical Report UCSC-CRL-87-27, (1987).

2. "A Model of Learning Based on the Principle of Cognitive Economy", X.
Xxxxxxxxxxxxx, Technical Report UCSC-CRL-88-33, (1988).

3. "Informed Parsimonious Inference of Prototypical Genetic Sequences", X.
Xxxxxxxxxxxxx, X. Xxxxxxxx, and X. Xxxxx, Proceedings of the Second
Workshop on Computational Learning Theory, (1989).

4. "Categorization of Macromolecular Sequences by Minimal Length Encoding", X.
Xxxxxxxxxxxxx, Ph.D. Thesis (1990).

5. "Reconstruction and Analysis of Human Alu Genes", X. Xxxxxxxxxxxxx, Journal
of Molecular Evolution, 32, 105-121 (1991).

6. "Enhanced Malignant Transformation Induced by Expression of a Distinct
Protein Domain of Ribonucleotide Reductase Large Subunit from Herpes
Simplex Virus Type 2", M., Ali, X. XxXxxxxx, X. Xxxxxxxxxxxxx, X. Xxxxx,
and X. Xxxxxxxxx, Proc. Natl. Acad. Sci., 88, 8257-8261 (1991).

7. "Prototypic Sequences for Human Repetitive DNA", X. Xxxxx, X. Xxxxxxxxxxxx,
and X. Xxxxxxxxxxxxx, J. Mol. Evol., 35, 286-291 (1992).

8. "Discovery by Minimal Length Encoding: A Case Study in Molecular
Evolution", X. Xxxxxxxxxxxxx and X. Xxxxx, Machine Learning Journal,
Special Issue on Machine Discovery, 12, nos. 1, 2, 3 (1993).

9. "Discovering Simple DNA Sequences by the Algorithmic Significance Method",
X. Xxxxxxxxxxxxx, and X. Xxxxx, Computer Applications in Biosciences, 9,
no. 4 (1994).

10. "Identification and Characterization of New Human Medium Reiteration
Frequency Repeats", X. Xxxxx, X.X. Xxxxxx, X.X. Xxxxxx, X. Xxxxxxxxxxxx, X.
Xxxxxxxxxxxxx, X. Xxxxxx, and X.X. Xxxxx, Nucleic Acids Research, 21, no.
5, 1273-1279 (1993).

11. "Discovering Sequence Similarity by the Algorithmic Significance Method",
X. Xxxxxxxxxxxxx, Proceedings of the First International Conference on
Intelligent Systems for Molecular Biology, (1993).

12. "Clone Clustering by Hybridization", X. Xxxxxxxxxxxxx, X. Xxxxxxxxx, X.
Xxxxxxxx, D. Grujuc, X. Xxxxxxxx, and X. Xxxxxxxxxxx, Genomics, in press.

13. "Repeat Analysis", X. Xxxxxxxxxxxxx, Chapter 13, Section IV, Handbook of
Genome Analysis, in press.

--------------------------------------------------------------------------------

Give the following information for the key personnel and consultants and
collaborators. Begin with the principal investigator/program director.
Photocopy this page for each person.

--------------------------------------------------------------------------------
NAME POSITION TITLE
Xxxxxx X. Xxxxxxx Director of Software Development

YEAR
INSTITUTION AND LOCATION DEGREE CONFERRED FIELD OF STUDY
-------------------------------------------------------------------------------------------------------------

California State University, Long Beach, CA B.S. 1983 Chemical
-------------------------------------------------------------------------------------------------------------

Professional Experience:
-----------------------

1980-1984 Research Associate
Specialty Labs Inc., Xxx Xxxxxxx, XX

0000-0000 Research Associate
Xxxxxxx Research Institute of the City of Xxxx, Xxxxxx, XX

0000-0000 Scientist/Systems Xxxxxxx
Xxxxxxx Xxxxxxxxxxx, Xxx., Xxxx, XX

0000-0000 Senior Software Engineer
Xxxxxxx Xxxxx, Xxx Xxxxx, XX

0000-0000 Software Consultant
Neurogen Corporation, Branford, CT

1995-Present Director of Software Development
CuraGen Corporation, Branford, CT

Honors and Awards:
-----------------

Gold Nugget Award, Associated Students Honors for Service, CSLUB, 1983
Chemistry Alumni Award, CSLUB, 1983
Distinguished Service Award, Department of Chemistry, CSLUB, 1983
Distinguished Service Award, School of Natural Science, CSLUB, 1983

Bibliography:
------------

1. "Evaluation of the recovery between samples presented in primary tubes and
those presented in cups on the Xxxxxxx Synchron CX(R)7", X.Xxxxx, and X.X.
Xxxxxxx, Clin.Chem. 37, 872 (1991).

2. "Differential detection of viral DNA and RNA in situ cells infected with
human cytomegalovirus", J.R. XxXxxxxx, X. X. Xxxxxxx, M. A. Xxxxxxxxx, and
X.X. Xxxx, Journal of Virological Methods 25, 301-314 (1989).

3. "Evaluation of Cold-Stable Rate-Xxxxx creatinine reagent for the use on the
Xxxxxxx Synchron CX4/5", X. X. Xxxxxxx et. al, Clin.Chem, 33 951 (1987).

--------------------------------------------------------------------------------

Give the following information for the key personnel and consultants and
collaborators. Begin with the principal investigator/program director.
Photocopy this page for each person.
--------------------------------------------------------------------------------
NAME POSITION TITLE

Xxxxxx X. Xxxxxxxxx Professor of Applied and Engineering Physics

--------------------------------------------------------------------------------
RESEARCH AND/OR PROFESSIONAL EXPERIENCE. Concluding with present position, list,
in chronological order, previous employment, experience and honors. Key
personnel include the principal investigator and any other individuals who
participate in the scientific development or execution of the project. Key
personnel typically will include all individuals with doctoral or other
professional degrees, but in some projects will include individuals at the
masters or baccalaureate level provided they contribute in a substantive way to
the scientific development or execution of the project. Include present
membership or any Federal Government public advisory committee. List, in
chronological order, the titles, all authors, and complete references to all
publications during the past three years and to representative earlier
publications pertinent to this application. DO NOT EXCEED TWO PAGES.

After completing his thesis in experimental physics at the end of 1979,
Xx. Xxxxxxxxx joined Xxxx Laboratories as a Member of Technical Staff in the
Device Physics Research Department. Research included studies of high-resolution
semiconductor fabrication processes including electron beam lithography with
high energy finely focused electron beams. This work resulted, at the time, in
the creation of the smallest wires and etched structures on GaAs. He did some
of the deepest UV lithography studies with excimer lasers.
At the breakup of AT&T, Xx. Xxxxxxxxx transferred to the newly created
Xxxx Communications Research and became a research manager in the Solid State
Science and Technology Laboratory. He formed and headed the group responsible
for advanced lithography and device fabrication and ultra-small experimental
device studies. His personal research centered on advanced semiconductor
processing and the properties of ultra-small structures. He explored ion etching
processes in compound semiconductors, with in several cases the first published
accounts of reactive ion etching processes in several material systems. Studies
of ion induced damage and enhanced compositional disordering were exploited to
create quantum wire and dot structures in GaAs/AlGaAs systems. He has worked
extensively on optical materials, device and system studies.
In 1989 Xx. Xxxxxxxxx joined the faculty of Cornell University as a
Professor in the School of Applied and Engineering Physics. From 1989 until 1995
he was Director of the National Nanofabrication Facility at Cornell University.
He is involved in collaborative projects on new molecular species for
microfabrication and cell growth and a developing project on the use of
microfabrication in biology.

Publications (last three years)

"Anisotropic reactive ion etching of InP in methane/hydrogen based plasmas",
X. X. XxXxxx, X. X. Xxxxxx, and X. X. Xxxxx, J. Vac, Sci. Technol, B. 9,
-
3535 (1991).

"Anisotropic reactive ion etching of InP in methane/hydrogen based plasmas",
X. XxXxxx, X. X. Xxxxxxxxx, and X. Xxxxxx, in Integrated Photonics Research,
1991, Technical Digest Series, (Optical Society of America, Washington, DC
1991), pp26-27.

"High resolution reactive ion etching of SiGe Alloys", X. X. Xxxxxxxxx, X. X.
Xxxxxxxxx, J. Vac, Sci. Technol, B. 11, 717 (1993).
--

"Lateral patterning of nanostructures", X. X. Xxxxxxxxx in Physics of
Nanostructure, A. Long, editor (1992) (NATO summer school).

"Ballistic electron emission microscopy investigation of SiGe nanostructures
fabricated using high resolution electron beam lithography and reactive ion
etching", X. Xxxxxx, X. X. Xxxxxxxxx, and X. X. Xxxxxxxxx, Appl. Phys. Lett. 61,
--
1040 (1992).

Principal Investigator/Program Director (Last, first, middle): Went, Xxxxxxx X.
--------------------------------------------------------------------------------

"Ballistic electron emission microscopy investigation of SiGe nanostructures
fabricated using reactive ion etching", X. X. Xxxxxxxxx, X. Xxxxxx, and X. X.
Xxxxxxxxx, J. Vac. Sci. Technol B, 10, 3112 (1992).
--

"A method of fabrication submicrometer gold wires on optical fibers", X. X.
Xxxxxxxxx, X. X. Xxxxxxxxx, and X. XxXxxx, Thin Silid Films, 208, 156 (1992).
---

"A self assembled monolayer electron beam resist on GaAs", X. X. Xxxxxxx, X. X.
Xxxxxxxxx, X. Xxxxxx, T. Xxx, X. X. Sheen and X. X. Xxxxxx, Appl. Phys. Lett,
62, 476, (1993).
--

"Proton implantation intermixing of GaAs/AlGaAs quantum xxxxx", X. X. Xxxxxxx,
X. X. Xxxxxxxxx and X. X. Xxxx, J. Appl. Physics 74, 3099, (1993).
--

"A simple surface emitting LED array for free-space optical interconnects",
H. F. Bare, X. Xxxx, D. A. Honey, X. Xxxxxxx, X. X. Xxxxxxxxx, X. Xxxx and
X. Xxxxx Photonics Technol. Lett, 5, 172 (1993).
-

"Fabrication of aspheric high numerical aperture reflective diffractive optic
elements using electron beam lithography", X. Xxxxxxx, X. Xxxxx, X. X.
Xxxxxxxxx, X. Xxxx, D. A. Honey and H. F. Bare, J. Vac. Sci. Technol. B, 12, 20
--
(1994).

"Photolithography of Porous Silicon", X. X. Xxxxxxxxx and X. X. Xxxxxxxxx, J.
Vac. Sci. Technol. B. 12, 161 (1994).
--

"Focused ion beam interactions with a shallow two-dimensional electron gas",
Y. A. Soh, X. X. Xxxxxx, X. X. Xxxxxxx, and X. X. Xxxxxxxxx, J. Vac. Sci.
Technol. B, 11, 2629 (1993).
--

"Self assembled monolayer electron beam resists on GaAs and SiO2", X. Xxxxxx, X.
X. Xxxxxxx, X. X. Xxxxxxxxx, X. X. Sheen, A. N. Xxxxxx, and X. X. Xxxxxx, J.
Vac. Sci. Technol. B, 11, 2823 (1993).
--

"Ballistic-electron-emission microscopy characteristics of reverse-biased
Schottky diodes", X. Xxxxxx and X. X. Xxxxxxxxx, Appl. Phys. Lett, 64, 2833
--
(1994).

"Fabrication of a multilevel structure for nanophysics in two-dimensional
electron gases", Y-A. Soh. X. X. Xxxxxx, X. X. Xxxxx, X. X. Xxxxxxxxx and
X. Xxxxxx, J. Vac. Sci. Technol. B, 12, 1372 (1994).
--

"Binary optics for optical interconnects", X. X. Xxxxx and X. X. Xxxxxxxxx, (to
appear in Proc. SPIE 2216 (1994))
----

"Scanning tunneling microscopy based lithography of octadecanethiol on Au and
GaAs", X. X. Xxxxxx X. X. Xxxxxxx, X. X. Xxxxxxxxx, X. X. Sheen and X. X.
Xxxxxx, Appl. Phys. Lett 65, 974 (1994).
--

"Diffractive phase elements based on two-dimensional artifical dielectrics",
F. T. Chen and X. X. Xxxxxxxxx, Optics Letters 20 No. 2, 121 (1995)
--

--------------------------------------------------------------------------------

Xxxxxx Xxxxxxx Research Scientist

--------------------------------------------------------------------------------
RESEARCH AND PROFESSIONAL EXPERIENCE: Concluding with present position, list in
chronological order previous employment, experience and honors. Key personnel
include the principal investigator and any other individuals who participate in
the scientific development or execution of the project. Key personnel typically
will include all individuals with doctoral or other professional degrees, but in
some projects will include individuals at the masters or baccalaureate level
provided they contribute in a substantive way to the scientific development or
execution of the project. Include present membership on any Federal Government
public advisory committee. List, in chronological order, the titles, all
authors, and complete references to all publications during the past three years
and to representative earlier publications pertinent to this application. DO NOT
EXCEED TWO PAGES.

Professional Experience:
------------------------
1989-1990 Research and Teaching Assistant, Center for Medical Research,
University of Sussex, United Kingdom.
1990-1992 Research Scientist, Medical Research Council, Cambridge, United
Kingdom.
1992-1993 Staff Scientist, Medical Research Council, Cambridge, United
Kingdom.
1993 Senior Scientist, Promega Corporation, Madison Wisconsin.
1993-present Research Scientist, Xxxxxxxxx Institute/MIT Center for Genomic
Research.

Publications:
-------------
Xxxxxxx, X.X., and Xxxxxxx, X.X.(1990). Automated fluorescent primer walking.
Technique-A journal of methods in cell and molecular biology. 2.6. 307-310.
Xxxxxxx, X.X., and Sulston, J.E.(1991). The resolution of compressions in
automated fluorescent sequencing. Nucl. Acids. Res. 19.10.2784.
Sulston, J., and Xxxxxxx, X.X., et al (1992). The C.elegans genome sequencing
project: A beginning. Nature 356:37-41.
Xxxxxxxx, R. and Xxxxxxx, X.X. et al (1992). A survey of expressed genes in
Caenorhabditis elegans. Nature genetics 1:114-123.
Xxxxxxx, X.X., Du, Z., Xxxxxxxx, N.D., and Xxxxxx, X.X.(1992). Fluorescence
chemistries for automated primer-directed DNA sequencing. Electrophoresis
13:552-559.
Xxxxxxx, X.X. (1992). M13 single-stranded purification using a biotinylated
probe and streptavidin coated magnetic beads. DNA sequence- J.DNA Sequencing
and Mapping. 3:65-69.
Xxxxxxx, X.X. The use of magnetic particles in molecular biology. In Automated
DNA sequencing and analysis techniques, Ed. Xxxxx Xxxxxx, X.
Xxxxxxxxx, R., and Xxxxxxx, X.X. et al. (1993). Sequencing the genome. WBG
93.340-343.

Berks, M., and Xxxxxxx, X.X. et al (1993). WBG 123.22-25.

Xxxxxx, A., Smaldon, N., Xxxxx, R., and Xxxxxxx, X.X. (1993). The Caenorhabdiris
elegans project: first steps in automation. Nature. 362:569-570.
Xxxxxx, R., Xxxxxxx, T., et al. (1994). 2.2Mb of contiguous nucleotide sequence
from chromosome III of C. elegans. Nature 368:32-38.
Xxxxxxx, X.X. (1994). In automated DNA sequence analysis. Ed. Xxxxx Xxxxxx,
Academic Press.

Xxxxxxx, X.X., X'Xxxxxx-Xxxxx, T., Xxx X., and Xxxxxxxxx, C. (1994). DNA
purification and isolation using a solid-phase, Nucl. Acids, Res. In press
Xxxxxxx, X.X., X'Xxxxxx-Xxxxx, T., Xxxxxxx, K., Xxxxxxxx, D., XxXxxxxxx, M.,
Xxxxxxxxx, C., and Xxxxx, C. (1994). Solid-phase molecular biology. Biomedical
products. December issue. Ed Xxxxx Xxxxx.

FF Principal Investigator/Program Director (Last, first, middle):
Went, Xxxxxxx X.
----------------
--------------------------------------------------------------------------------
Page 2

Xxxxxxx, X.X., Banerjee S.R., Brodowski, C., Days, F., Xxxxxxxx, D., and
Xxxxxxx, K. (1995) Thermal Cycle DNA Sequence set up using a modified
lab workstation. In press.
Xxxxxxxxx, X.X., Xxxxxxxxxx, X.X., Sulston, J., and Xxxxxxx, X.X., 41 Kilobases
of Analyzed Sequence from the pseudoautosomal and Sex-determining
regions of the short arm of Human Y chromosome. Submitted.
Xxxxxxxxx, C., Xxx, A., and Xxxxxxx, X.X., Isolation of DNA from agarose gel
using a solid-phase. Submitted.

XxXxxxxxx, M., and Xxxxxxx, X.X., Solid-Phase Reverse immobilzation for the
isolation of PCR products. Submitted.
Xxxxxxx, K., Xxxxxxxx, D., and Xxxxxxx, X.X., Solid-Phase Reversible
immobilization for the isolation of cosmid DNA suitable for library
construction. Submitted.

--------------------------------------------------------------------------------

Xxxxxxx X. Xxxxxx Vice President, Research

North Carolina State University B.S. 1985 Chem. Eng.

University of California, Berkeley Ph.D. 1990 Chem. Eng.

--------------------------------------------------------------------------------
RESEARCH AND PROFESSIONAL EXPERIENCE. Concluding with present position, list, in
chronological order, previous employment, experience and honors. Key personnel
include the principal investigator and any other individuals who participate in
the scientific development or execution of the project. Key personnel typically
will include all individuals with doctoral or other professional degrees, but in
some projects will include individuals at the masters or baccalaureate level
provided they contribute in a substantive way to the scientific development or
execution of the project. Include present membership on any Federal Government
public advisory committee. List, in chronological order, the titles, all
authors, and complete references to all publications during the past three years
and to representative earlier publications pertinent to this application. If
the list of publications in the last three years exceeds two pages, select the
most pertinent publications. DO NOT EXCEED TWO PAGES.

Experience:
-----------

1985-1990 Graduate Research Assistant, University of California, Berkeley
1990 Postdoctoral Research Fellow, University of California, Berkeley
1990-1993 Senior Research Engineer, Air Products and Chemicals, Inc.,
Allentown, PA
1993-present Vice President, Research, Soane BioSciences, Inc.

Awards:
-------
Principal Investigator on NIH SBIR awards and Advanced Technology Program award.

Publications:
-------------
Sassi, A.P., X. Xxxxxxx, X.X. Xxxxxx, X.X. Xxxxxx and X.X. Xxxxxxxxx, Xxxxx
Carlo Simulations of Hydrophobic Weak Polyelectrolytes; Titration Properties
and pH-Induced Structural Transitions. J. Chem. Phys., 97(11), 8767(1992).
Xxxxx, X.X., X.X. Xxxxxx, X.X. Xxxxxx and X.X. Xxxxxxxxx, Swelling Equilibria
for Weakly-Ionizable, Temperature-Sensitive Hydrogels, Macromolecules, 24,
549(1991).
Xxxxxx, X.X., X. Xxxxxxx, X.X. Xxxxx, X.X. Xxxxxx and X.X. Xxxxxxxxx, Xxxxx
Carlo Simulations of Hydrophobic Polyelectrolytes: Evidence for a Structural
Transition in Response to Increasing Chain Ionization, J. Chem. Phys., 93(4),
2715(1990).
Xxxxxx, X.X., X.X. Xxxxxx and X.X. Xxxxxxxxx, Configurational Properties of
Partially Ionized Polyelectrolytes from Monte Carlo Simulation,
Macromolecules, (1990).
Xxxxxxx, S., X.X. Xxxxxx, X.X. Xxxxxx and X.X. Xxxxxxxxx, Swelling Equilibria
for Ionized Temperature Sensitive Gels in Water and Aqueous Salt Solutions, J.
Chem. Phys., 92(3), 2061(1990).
Xxxxxx, X.X., X.X. Xxxxxx, X.X. Xxxxxxxxx, Molecular Thermodynamics of Aqueous
Polymers and Gels, in Absorbent Polymer Technology, Elsevier Publishers
(1990).
Xxxxxx, X.X., Swelling Equilibria for Positively Ionized Polyacrylamide
Hydrogels, Macromolecules, 23, 1096(1990).
Xxxxxx, X.X., X.X. Xxxxxx and X.X. Xxxxxxxxx, Thermodynamics of Aqueous Systems
Containing Hydrophilic Polymers or Gels, AICHE J., 35(5), 803(1989).
Michel, S., X.X. Xxxxxx and X.X. Xxxxxxxxx, Mutual Solubilities of Water and
Hydrocarbons from an Equation of State, Fluid Phase Equil., 45, 173(1989).

Xxxxxx, X.X., X. Xxxxxx and X.X. Xxxxxxxxx. Correlation of Liquid-Liquid
Equilibria for Some Water/Organic-Liquid Systems in the Region 20-250C. Ind.
Eng. Chem. Res., 27(11), 2182(1988).
Xxxxxx, X.X., X. Xxxxxx and X.X. Xxxxxxxxx. High-Temperature Mutual
Solubilities for some Binary and Ternary Aqueous Mixtures Containing
Aromatic and Chlorinated Hydrocarbons, J. Chem. Eng. Data. 33(4), 502
(1988).

Patents:
--------
X.X. Xxxxxx, X. Xxxxxxx, X.X. Xxxxx and X.X. Xxx. Separation Media for
Electrophoresis, patent pending.

--------------------------------------------------------------------------------

Xxxxx X. Xxxxx Chairman, Scientific Advisory Board

National Taiwan University B.S. 1973 Chemistry
University of California, Berkeley M.S. 1977 Chemical Eng.
University of California, Berkeley Ph.D. 1978 Chemical Eng.

--------------------------------------------------------------------------------
RESEARCH AND PROFESSIONAL EXPERIENCE. Concluding with present position, list, in
chronological order, previous employment, experience and honors. Key personnel
include the principal investigator and any other individuals who participate in
the scientific development or execution of the proper Key personnel typically
will include all individuals with doctoral or other professional degrees, but in
some projects will include individuals at the masters or baccalaureate level
provided they contribute in a substantive way to the scientific development or
execution of the project. Include present membership or any Federal Government
public advisory committee. List, in chronological order, the titles, all
authors, and complete references to all publications during the past three years
and to representative earlier publications pertinent to this application. If
the list of publications in the last three years exceeds two pages, select the
most pertinent publications. DO NOT EXCEED TWO PAGES.

Experience
----------

1978-1979 Postdoctoral Research Associate, Department of Chemical
Engineering, UC Berkeley
1979-1983 Assistant Professor, Department of Chemical Engineering, UC
Berkeley
1983-1987 Associate Professor, Department of Chemical Engineering, UC
Berkeley
1984-present Associate Faculty Scientist, Center for Advanced Materials,
Xxxxxxxx Berkeley Lab
1985-1986 Sabbatical Leave at IBM, Almaden Research Laboratory and Kyoto
University, Japan
1986-1989 Vice Chairman, Department of Chemical Engineering, UC Berkley
1987-1994 Professor, Department of Chemical Engineering, UC Berkeley
1994-present Adjunct Professor, Department of Chemical Engineering, UC
Berkeley
1991-present Founder and Chairman, Soane Technologies, Inc.
1993-present Chairman, Scientific Advisory Board, Soane BioSciences, Inc.

Honors:
------
Xxxx X. Xxxxxxx Fellowship, 1976
Xxxxxxx and Xxxxx Xxxxxxx Teach-Scholar Award, 1984

Society Activities:
------------------
Editorial Advisory Board, Journal of Applied Polymer Science, 1983-1989
Editorial Advisory Board, Journal of Biomaterials, 1985-present
Membership and Examination Committees, Polymer Division, American Chemical
Society, 1982-present
Board of Directors, Golden Gate Chapter, Society of Plastics Engineers,
1982-present
Executive Committee, Northern California Section, American Institute of
Chemical Engineers, 1983-1984

Selected Publications (total greater than 170):
----------------------------------------------
Xxxxxx, X.X., X.X. Xxxxxx and X.X. Xxxxx. A Transient Entanglement Coupling
Mechanism for DNA Separation by Capillary Electrophoresis in Ultra-Dilute
Polymer Solutions.
Electrophoresis. 15.597 (1994).

--------------------------------------------------------------------------------

Xxxxxxxx, X.X., A Mathematical Model for Planarization of Microelectronic
Topographies. J. Electochem. Soc., 141,585 (1994)
Monk, D.J., D.S. Soane and X.X. Xxxx, Hydrofluoric Acid Etching of Silicon
Dioxide Sacrificial Layers, 2. Modeling, J. Electochem. Soc., 141, 270
(1994).
Monk, D.J., D.S. Soane and X.X. Xxxx, Hydrofluoric Acid Etching of Silicon
Dioxide Sacrificial Layers, 1. Experimental Observations, J. Electrochem,
Soc., 141. 264 (1993).
Xxxxxx, A.E.D.S. Soane and X.X. Xxxxxx, Capillary Electrophoresis of DNA in
Uncrosslinked Polymer Solutions, J. Chrom., 652.3 (1993).
Bae, Y.C., and D.S. Soane, Polymeric Separation Media for Electrophoresis;
Crosslinked Systems or Entangled Solutions, J. Chrom. A., 652 (1), 17
(1993).
Monk, D.J., D.S. Soane and X.X. Xxxx, A Review of the Chemical Reaction
Mechanism and Kinetics for Hydrofluoric Acid Etching of Silicon Dioxide for
Surface Micromachining Applications, Thin Solid Films, 231,1 (1993).
Monk, X.X. Xxxxx and X.X. Xxxx, LPCVD Silicon Dioxide Sacrificial Layer Etching
for Surface Micromachining, Proc. Mac. Res. Soc. 264,303 (1992).
Hino, T., X.X. Xxxxxxxx and D.S. Soane, Dynamic Light Scattering Studies of HEC
Solutions used as Sieving Media for Electrophoretic Separations, J. Chrom.,
608,79 (1992).
Xxxxxxxx, X.X., and X.X. Xxxxx, Capillary Electrophoresis of DNA in Entangled
Polymer Solutions, J. Chrom., 559,257 (1991).
Xxxxxxxx, X.X., and D.S. Soane, Experimental and Theoretical Studies of DNA
Separations by Capillary Electrophoresis in Entangled Polymer Solutions,
Biopolymers, 31,1221 (1991).

Selected Presentations:
-----------------------
Soane, D.S., Advances in Gel Matrices for DNA Electrophoresis, presented at The
Human Genome Project: Commercial Implications, San Francisco, CA. March,
1995.
Soane, D.S., Replaceable Crosslinked Gels for DNA Analysis by Capillary
Electrophoresis, presented at Third International Symposium on Capillary
Electrophoresis, York, UK, August, 1994.

Books:
------
Soane, D.S. ed. Polymers for Biotechnology; Macromolecular Separation and
Identification. Prentice Hall, New York, 1992.
Soane, D.S. and X. Xxxxxxxxxx, Polymers in Microelectronics; Fundamentals and
Applications, Elsevier, New York and Amsterdam, 1989.

Selected Patents (total greater than 15);
-----------------------------------------
Soane, D.S. and X.X. Xxx, Separation Media for Electrophoresis, WO 94/10561.
Soane, D.S., Mosaic Microcolumns, Slabs and Separation Media for Electrophoresis
and Chromatography, US Patent 5,135,627 (1992).
Soane, D.S. and Z.M. Soane, Method and Device for Moving Molecules by the
Applications of a Plurality of Electrical Fields, US Patent 5.126.022
(1992).
Soane, D.S. Controlled Casting of a Shrinkable Material, US Patent 5.110.514
(1992).
Soane, D.S., Gel Casting Method and Apparatus, US Patent 5.061.336 (1991).
Soane, D.S., Casting of Gradient Xxxx, US Patent 5.135.627 (1991).

Xxxxxxx Xxxxxx Amigo Senior Scientist

--------------------------------------------------------------------------------
EDUCATION (Begin with baccalaureate or other initial professional education,
such as nursing, and include postdoctoral training.
--------------------------------------------------------------------------------
YEAR
INSTITUTION AND LOCATION DEGREE CONFERRED FIELD OF STUDY
--------------------------------------------------------------------------------
University of Valladolid, Spain BS 0000 Xxxxxxxxx
Xxxxxxxxxx xx Xxxxxxx, Xxxxxxxx MSEC 0000 Xxxxxxx & Xxxx.
Xxxxxxxxxx xx Xxxxxxx, Xxxxxxxx Ph.D. 1988 Macromolecular
Ch.
--------------------------------------------------------------------------------
RESEARCH AND PROFESSIONAL EXPERIENCE. Concluding with present position, list, in
chronological order, previous employment, experience and honors. Key personnel
include the principal investigator and any other individuals who participate in
the scientific development or execution of the project Key personnel typically
will include all individuals with doctoral or other professional degrees, but in
some projects will include individuals at the masters or baccalaureate level
provided they contribute in a substantive way to the scientific development or
execution of the project. Include present membership on any Federal Government
public advisory committee. List, in chronological order, the titles, all
authors, and complete references to all publications during the past three years
and to representative earlier publications pertinent to this application. If
the list of publications in the last three years exceeds two pages, select the
most pertinent publications. DO NOT EXCEED TWO PAGES.

Experience:
-----------
1979-1981 Science Teacher, ICEDE Professional School, Burgos, Spain
1981-1984 Research Assistant, Polymer Technologies Inc., University of
Detroit, Xxxxxxx, XX
0000-0000 Graduate Research Assistant, Xxxxxxxxxx xx Xxxxxxx, Xxxxxxx, XX
0000-0000 Materials Scientist, Polymer R&D, Xxxxxxxxxx Xxxxxx Hind,
Xxxxxxxxx, XX
0000-0000 Manager, Analytical Chemistry R&D, Xxxxxxxxxx Xxxxxx Hind,
Sunnyvale, CA
1994 Adjunct Professor, Xxx Xxxx Xxxxx Xxxxxxxxxx, Xxx Xxxx, XX
0000-0000 Manager, Polymer R&D, Xxxxxxxxxx Xxxxxx Hind, Sunnyvale, CA
1995-present Senior Scientist, Soane BioSciences, Inc.

Publications:
-------------

X.X. Xxxxxx-Amigo and X. Xxxxxxx, ESR of Cu/2+/ in Nafion Ionomers,
Polymer Preprints (American Chemical Society, Polymer Division
Preprints, 27,337(1986).

X.X. Xxxxxx-Amigo and X. Xxxxxxx, Detection of Isolated and Paired
Cu/2+/ Ions in Nafion Membranes by ESR Spectroscopy, Journal of Physical
Chemistry, 90, 6353 (1986).

X. Xxxxxx, X. Xxxxx, M.D. Xxxxxxx, X.X. Xxxxxx-Amigo and X. Xxxxxxx, An
Electron Spin Resonance Investigation of the Motion of Lipid Peroxyl
Radicals, Journal of Physical Chemistry. 91, 492, (1987).

X.X. Xxxxxx-Amigo and X. Xxxxxxx, Study of Mobile and Rgid Peroxy
Radicals in Polypropylene, Macromolecules, 20, 795, (1987).

X.X. Xxxxxx-Amigo and X. Xxxxxxx, Binding of Paramagnetic Ions in
Ionomers, Cu/2+/ and Ti/3/- in Nafion Membranes, Journal of Chemical
Society, Faraday Transactions I. 83, 3575 (1987).

X.X. Xxxxxx-Amigo and X. Xxxxxxx, Detection of Isolated, Paired and
Clustered Ti/3+/ in Nafion by ESR Spectroscopy, Polymer Preprints
(American Chemical Society, Polymer Division Preprints), 28(2), 363,
(1987).

X. Xxxxxxx, X.X. Xxxxxx, X.X. Xxxxxx-Amigo and X. Xxxxxxxx, Study of
Phase Separation in LPNs Using Nitroxide Spin Labels, Macromolecules,
22, 822 (1989).

X.X. Xxxxxx-Amigo and X. Xxxxxxx, Determination of the Distance Between
Paramagnetic Centers from ESR Spectra at L. S and X Bands, Cu/2+/ in
Nafion Ionomers. Macromolecules, 22 2638 (1989).

--------------------------------------------------------------------------------

FF Principal Investigator Program Director (Last, first, middle):
Went Xxxxxxx X.
---------------
--------------------------------------------------------------------------------
Biographical Sketch
Xxxxxxx X. Amigo
Page 2

X.X. Xxxxxx-Amigo and X. Xxxxxxx, Local Environment and Clustering of
Cations in Ionomers. ESR of Cu/2/+ in Nafion Swollen by Water,
Methanol,Dimethylformamide and Tetrahydrofuran. Macromolecules, 22, 2634
(1989).

X. Xxxxxxx, X.X. Xxxxxx-Amigo and X.X. Xxxxx, Structure of Cu/2/+_Cu/2/
Dimers in Nafion Swollen by Water, Methanol, DMF and THF. ESR Results and
Theoretical Simulations. Journel of Physical Chemistry, 93, 7906 (1989)

X. Xxxxxxx and X.X. Xxxxxx-Amigo, Co-Cation Enhanced Electron Transfer in
Nafion Neutralized by Ti/3/+. Study of O\\2\\ Formation by ESR. Polymer
Preprints (American Chemical Society), 30 (1), 440 (1989).

X.X. Xxxxxx-Amigo and X. Xxxxxxx, O\\2\\ Formation in Nafion by Electron
Transfer from Ti/3/+. The Effect of Al/3/+ as Cocation, Journal of Physical
Chemistry, 93, 7526 (1989).

Xxxxxxxxx Xxxxxxx, X.X. Xxxxxx-Amigo and Xxxxxx Bednareck, Multifrequency
Electron Spin Resonance and Electron Nuclear Double Resonance Studies of
Metal Carions in Perfluorinated Ionomers, Colloids and Surfaces A:
Physicochemical and Engineering Aspects. 72, 1 (1993).

Xxxxxxxxx X. Xxxxx Senior Research Engineer

University of Washington, Seattle BSChE 1988 Chemical Eng.

University of California, Berkeley Ph.D. 1994 Chemical Eng.

--------------------------------------------------------------------------------
RESEARCH AND PROFESSIONAL EXPERIENCE. Concluding with present position, list, in
chronological order, previous employment, experience and honors. Key personnel
include the principal investigator and any other individuals who participate in
the scientific development or execution of the project Key personnel typically
will include all individuals with doctoral or other professional degrees, but in
some projects will include individuals at the masters or baccalaureate level
provided they contribute in a substantive way to the scientific development or
execution of the project. Include present membership on any Federal Government
public advisory committee. List, in chronological order, the titles, all
authors, and complete references to all publications during the past three years
and to representative earlier publications pertinent to this application. If the
list of publications in the last three years exceeds two pages, select the most
pertinent publications. DO NOT EXCEED TWO PAGES.

Experience:
-----------
1988 Engineer Intern, Exxon Production Research, Xxxxxxx, XX
0000-0000 Graduate Research Assistant, University of California, Berkeley
1994 Visiting Researcher, Eisai Co., Ltd., Tsukuba, Japan
1995-present Senior Research Engineer, Soane BioSciences, Inc.

Awards:
-------
Principal Investigator on NIH SBIR Phase I grant, Constant-Field Electrophoresis
of Large DNA.

Publications:
-------------

Sassi, A.P., X.X. Xxxxxx, and X.X. Xxxxxxxxx, Phase Equilibria for Aqueous
Protein/Polyelectrolyte Gel Systems, submitted to AIChE Journal.
Sassi, A.P., X. Xxxxx, X.X. Xxxxxx, and X.X. Xxxxxxxxx, Partitioning of
Hexavalent Chromium in Temperature-Sensitive, Polyelectrolyte Hydrogels,
submitted to Polymer Gels and Networks.
Sassi, A.P., X. Xxxx, X. Xxx, X.X. Xxxxxx, and X.X. Xxxxxxxxx, Partitioning of
Proteins and Small Biomolecules in Temperature-and pH-Sensitive Hydrogels,
accepted by Polymer (1995).
Sassi, A.P. S.H. Xxx, Y. Park, X.X. Xxxxxx, and X.X. Xxxxxxxxx, Sorption of
Lysozyme by HEMA Copolymer Hydrogels, accepted by Journal of Applied Polymer
Science (1995).
Sassi, A.P., X.X. Xxxxxx, and X.X. Xxxxxxxxx, Characterization of Size-Exclusion
Effects in Highly Swollen Hydrogels: Correlation and Prediction, accepted by
Journal of Applied Polymer Science (1995).
Sassi, A.P., X.X. Xxxxxx, and X.X. Xxxxxxxxx, Feasibility Studies for
Separations Processes Using Environmentally Sensitive Hydrogels, Lawrence
Berkeley Laboratory, LBL Report #36405 (1994).
Sassi, A.P., J.L.F. Abascal, H.W. Blanch, and J.M. Prausnitz, Monte Carlo
Simulations of a Hydrophobic Weak Polyelectrolyte. Charge Distribution as a
Function of Conformation. J. Chem. Phys. 99(5), 4231 (1993).

FF Principal Investigator Program Director (Last, first, middle):
Went Gregory T.
---------------
--------------------------------------------------------------------------------
Biographical Sketch
Alexander P. Sassi
Page 2

Sassi, A.P., S. Beltran, H.H. Hooper, H.W. Blanch, J.M. Prausnitz and R.A.
Siegel Monte Carlo Simulations of Hydrophobic Weak Polyelectrolytes:
Titration Properties and pH-Induced Structural Transitions for Polymers
Containing Weak Electrolytes, J. Chem. Pys., 97 (11), 8767 (1992).

Sassi, A.P., H.W. Blanch, and J.M. Prausnitz, Crosslinked Gels as Water
Absorbents in Separations, chapter 8 in Polymer Applications for
------------------------
Biotechnology, D.S. Soane, ed. Englewood Cliffs: Prentice Hall (1992)
-------------

Hooper, H.H. S. Beltran. A.P. Sassi, H.W. Blanch and J.M. Prausnitz, Monte Carlo
Simulations of Hydrophobic Polyelectrolytes: Evidence for a Structural
Transition In Response to Increasing Chain Ionization, J. Chem. Phys.,
93 (4), 2715 (1990).

Danny Hion Research Biochemist

--------------------------------------------------------------------------------
EDUCATION (Begin with baccalaureate or other initial professional education,
such as nursing, and include postdoctoral training.
--------------------------------------------------------------------------------
YEAR
INSTITUTION AND LOCATION DEGREE CONFERRED FIELD OF STUDY
--------------------------------------------------------------------------------
University of California, Berkeley BA 1992 Molecular
Biology
Genetics
--------------------------------------------------------------------------------
RESEARCH AND PROFESSIONAL EXPERIENCE. Concluding with present position, list, in
chronological order, previous employment, experience and honors. Key personnel
include the principal investigator and any other individuals who participate in
the scientific development or execution of the project Key personnel typically
will include all individuals with doctoral or other professional degrees, but in
some projects will include individuals at the masters or baccalaureate level
provided they contribute in a substantive way to the scientific development or
execution of the project. Include present membership on any Federal Government
public advisory committee. List, in chronological order, the titles, all
authors, and complete references to all publications during the past three years
and to representative earlier publications pertinent to this application. If the
list of publications in the last three years exceeds two pages, select the most
pertinent publications. DO NOT EXCEED TWO PAGES.

Experience:
-----------
1993-present Research Biochemist, Soane BioSciences, Inc.
Experienced in DNA sequencing sample prep and electrophoresis
(Slab and Capillary).

Give the following information for the key personnel and consultants and
collaborators. Begin with the principal investigator/program director.
Photocopy this page for each person
--------------------------------------------------------------------------------
NAME POSITION TITLE

Shi Lin Postdoctoral Scientist

--------------------------------------------------------------------------------
EDUCATION (Begin with baccalaureate or other initial professional education,
such as nursing, and include postdoctoral training
--------------------------------------------------------------------------------
YEAR
INSTITUTION AND LOCATION DEGREE CONFERRED FIELD OF STUDY
--------------------------------------------------------------------------------
South China University of Technology BS 1982 Chem. Eng.
South China University of Technology MS 1985 Chem. Eng.
SUNY College of Env. Sci. & Forestry Ph.D. 1994 Polym. Chem
Syracuse
--------------------------------------------------------------------------------
RESEARCH AND PROFESSIONAL EXPERIENCE. Concluding with present position, list, in
chronological order, previous employment, experience and honors. Key personnel
include the principal investigator and any other individuals who participate in
the scientific development or execution of the project. Key personnel typically
will include all individuals with doctoral or other professional degrees, but in
some projects will include individuals at the masters or baccalaureate level
provided they contribute in a substantive way to the scientific development or
execution of the project. Include present membership on any Federal Government
public advisory committee. List, in chronological order, the titles, all
authors, and complete references to all publications during the past three years
and to representative earlier publications pertinent to this application. If the
list of publications in the last three years exceeds two pages, select the most
pertinent publications. DO NOT EXCEED TWO PAGES.

Experience:
-----------
1982 Research Associate, Guangdong Provincial Research Institute of
Man-Made Fiber, China
1982-1985 Graduate Assistant, South China University of Technology
1985-1986 Faculty of Chemical Engineering, South China University of
Technology
1987-1994 Research Project Assistant, SUNY College of Environmental Science
and Forestry, Syracuse
1995-present Postdoctoral Scientist, Soane BioSciences, Inc.

Publications:
-------------
Shi Lin and Israel Cabasso, Synthesis and Characterization of
Carbamoylphosphonate Monomer and It's Copolymer with Styrene, II, submitted
to J. Polym. Sci. Polym. Chem. Ed.
Shi Lin and Israel Cabasso, Synthesis and Properties of
Poly{5-oxy[2-(2',2'-diethoxyphosphonoethyl)-1.3-dioxane]methyl siloxane}, to
be published
Shi Lin and Israel Cabasso, Synthesis and Characterization of
Poly{[(diethylphosphonobenzyl) a. b-ethyl] methyl siloxane} and Its Salt
Complexes, to be published
Su Maoyao, Gao Guang and Lin Shi, A Study on the Effects of Pretreatment by
Chemical Activation on the Fine Structure and Reactivity of Cellulose Fiber,
J. South China University of Technology, 17(1), 94, (1984)
Yang ZhiLi, Wu GuoMin, Mei ChienFong, Gao Guang, Lin Shi, Liu HaiMin and Zou
JingHong, Study on the Manufacture of Rayon Fiber from a PF/DMSO Solvent
System, Cellulose Chem. Technol., 21.493 (1987)

Patents:
--------
I. Cabasso and S. Lin, Water Soluble Phosphorylated Polysiloxanes. US patent
pending.
I. Cabasso and S. Lin, Hydrogels and Salt Complexes form Novel Phosphorylated
Siloxane. US patent pending.

--------------------------------------------------------------------------------

William Lee Research Specialist

Massachusetts Institute of Technology B.S. 1995 Mechanical Eng.

--------------------------------------------------------------------------------
RESEARCH AND/OR PROFESSIONAL EXPERIENCE: Concluding with present position, list
in chronological order previous employment, experience, and honors. Key
personnel include the principal investigator and any other individuals who
participate in the scientific development or execution of the project. Key
personnel typically will include all individuals with doctoral or other
professional degrees, but in some projects will include individuals at the
masters or baccalaureate level provided they contribute in a substantive way to
the scientific development or execution of the project. Include present
membership on any Federal Government public advisory committee. List, in
chronological order, the titles, all authors, and complete references to all
publications during the past three years and to representative earlier
publications pertinent to this application. DO NOT EXCEED TWO PAGES.

Professional Experience:
------------------------

1992-1995 Artificial Muscle Group. Artificial Intelligence Laboratory,
MIT
(1995) Second Phase Gel Model
(1994) pH Imaging System
(1994) Linear Gel Based Actuaor Test Bed
(1994) Small Scale Test Bed and Gel Fluid Model
(1993) Tabletop Cylindrical Gel Based Actuator Test Bed
(1992) Investigation of Mechanical Properties of PAN Fiber Gels
1995-present Research Specialist. Whitehead Institute for Biomedical
Research/MIT Center for Genome Research

Publications:
-------------
None.

Lincoln Stein Research Scientist

--------------------------------------------------------------------------------
RESEARCH AND/OR PROFESSIONAL EXPERIENCE: Concluding with present position, list,
in chronological order, previous employment, experience and honors, the
principal investigator and any other individuals who participate in the
scientific development or execution of the project. Key personnel typically will
include all individuals with doctoral or other professional degrees, but in some
projects will include individuals at the masters or baccalaureate level provided
they contribute in a substantive way to the scientific development or execution
of the project. Include present membership on any Federal Government public
advisory committee. List, in chronological order, the titles, all authors,
during the past three years and to representative earlier publications pertinent
to this application. DO NOT EXCEED TWO PAGES.

Professional Experience:
------------------------
1980-1982 Graduate Student Laboratory of George Scangos, John Hopkins
University Dept. of Biology; eukaryotic transcription promoters
using DNA-mediated gene transfer
1983 Graduate Student Laboratory of Richard Mulligan, M.I.T. Center
for Cancer Research; extendin of retrovirus vectors
1986 Microbiology laboratory instructor, New England Baptist Hospital
School of Nursing
1986-1988 Lecturer for veterinary parasitology and infectious disease
courses at Tufts University Veterinary
1984-1989 Graduate Student Laboratory of John David Harvard School of
Public Health; cloning of deve?? regulated genes from
Schistosoma mansoni
1990 Instructor for Harvard Medical School courses in infectious
diseases and gastrointestinal pathop???
1992-1994 Assistant Director, Informatics Core, Whitehead Institute/MIT
Center for Genome Research.
1990-present Instructor, Harvard Medical School, Department of Pathology.
Brigham and Women's Hospital
1994-present Director, Informatics Core, Whitehead Institute/MIT Center for
Genome Research.

Honors and Awards:
------------------

1978 National Merit Scholarship, Johns Hopkins University
1979 Phi Beta Kappa, Johns Hopkins University
1984 Medical Scientist Training Program Scholarship, Harvard Medical
School
Publications:
-------------

Copeland, N.G., D.J. Gilbert, N.A. Jenkins, J.H. Nadeau, J.T. Eppig, L.J.
Maltais, J.C. Miller, W.F. Dietrich, S.E. Lincoln, A. Weaver, D.C. Joyce, M.
Merchant, M. Wessel, H. Katz, L.D. Stein, M.P. Reeve, R.D. Dredge, A. Marquis,
N. Goodman, and E.S. Lander(1993) Genome Maps IV Science 262:67-82
--------------
Copeland, N.G., N.A. Jenkins, D.J. Gilbert, J.T. Eppig, L.J. Maltais, J.C.
Miller, W.F. Dietrich, A. Weaver, R.G. Steen, L.D. Stein, J.H. Nadeau, and
E.S. Lander(1993) A genetic linkage map of the mouse: C applications and
future prospects. Science 262:57-66.
-------
Dietrich, W.F., J.C. Miller, R.G. Steen, M. Merchant, D. Damron, R. Nahf, D.C.
Joyce, R.D. Dredge, A. Weaver, L.D. Stein, N. Goodman, D.C. Page, and E.S.
Lander(1994). A genetic map of the mouse with 4,006 simple length
polymorphisms. Nature Genetics, 7:220-245.
------ --------
Genest, D., Stein, L., Cibas, E., Sheets, E., Zitz. J., Crum, C.(1993) A Binary
(Bethesday) System for Class Cancer Precursors: Criteria, Reproducibility and
Viral Correlates. Human Path, 24:730-736.
----------
Goodman, N., Rozen S., Stein, L.(1993). Requirements for a Deductive Query
Language in the MapBase Ge???? Database. Workshop on Programming with Logic
----------------------------------
Databases, Vancouver, BC. Oct. 30, 1993.
---------
Goodman, N., Rozen S., Stein, L.(1994). Constructing a domain-specific DBMS
using a persistent object systems International Workshop on Persistent Object
-------------------------------------------
Systems, In press.
-------

FF Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------
Page 2
Miller, J., W. Dietrich, R. Steen, D. Joyce, M. Merchant, M. Wessel, D. Damron,
R. Nahi, L. Stein, R. Dredg M. Daly, M.P. Reeve, N. Goodman, C.J. Lord,
C.T. Montague, J.B. Prins. J. Todd, and E.s. Lande SSLP/Microsatellite
genetic linkage map of the mouse. In: Genetic Variants and Strains of the
------- -------- --- ------- -- ---
Laboratory Edition. M.F. Lyon, and A.G. searle, eds., Oxford University
---------- -------
Press.
Shoemaker, C. Ramachandran, H. Landa, H. dos Reis, M. Stin, L. (1992),
Alternative splicing of the Six gene encoding a homologue of epidermal
growth factor receptor. Mol Biochem Parasitol 53:17-32.
---------------------
Skelly, P.J., Stein, L.D. Shoemaker, C. (1993). Expression of Schistosoma
mansoni genes involved in ____ oxidative glucose metabolism during the
cercaria to adult transformation. Mol Biochem Parasitol. 60:93
STEIN, L. SNYDR-MICHAL, J., AND GREENES, R. (1991) Realistic Viewing and
Manipulation of Radiographic ____ Computer--a User Interface for
Educational Applications. Journal of Digital Imaging 4:3 169-176.
Stein, L., Ham. D., and David J. (1990), A cloned ATP:Guanidino Kinase in the
Trematode Schistosoma ___ Duplicated Structure, J. Biol. Chem. 265:6582-
--------------
6588.
Stein, L., David, J. (1987), Isolation of a tandemly repeated cDNA from S.
mansoni which contains four long reading frames. Federation Proc., April
----------------
1987.
Stein L., David, J. (1986). Cloning of a developmentally regulated tegument
antigen of Schistosoma mansoni: 20:253-264
Stein, L., Dessein. A., David. J. (1985). Cloning of two Schistosome Antigens
Recognized by Human _______. Proc. April. 1985.
----
Stein. L., Dessein. A., Harn. D., Miller. J., Bina. J., David. J. (1984).
Immunoprecipitation of Schistosome _____ Translation Products using Human
Antisera. Federation Proc., April. 1984.
---------------
Stein. L., Marquis> A., Dredge. E., Reeve. M.P., Daly. M., Rozen. S., Goodman.
N. (1994). Splicing UNIX Mapping Laboratory. USENIX Summer Technical
Conference. pp. 221-229. 1994.
Samuelson. J., and Stein. L. (1989). Schistosoma mansoni: increasing saline
concentration signals cercariae to schistosomula. Exp. Parasitol. 69:23-29.
--------------
Stein. L., Rozen. S., and Goodman. N. (1995). Managing laboratory Workflow With
LabBase. In Proceedings of the 1994 Conference on Computers in Medicine
(CompMed94). World Scientific Publishing Company.
Stein. L. (1995). How to Set Up and Maintain a World Wide Web Site: the Guide
for Information Providers. Addison-Wesley Publishing. Reading. MA.

Software Development

Explorer (1987) a medical hypertext teaching system developed in the laboratory
of Robert Greenes at Harvard ______
RWS (1987) an ultrasound image analysis package used by the Framingham Heart
Study to asses carotid _______ disease.
CWS (1990) an ultrasound image acquisition and analysis package based loosely on
RWS and used by the ______ Cardiovascular Health and the IRAS studies.
Quote Init (1988) a desktop publishing utility for producing typographically
correct quotes.
QT Batch Compressor (1991) an image compression utility for the Macintosh.
MapBase. and LabBase (1992.1994) two object-oriented genome databases for UNIX
platforms.
RHMAPPER (1994) a package for creating radiation hybrid maps for genome mapping.
XXX.xx (1995) An interface between the World Wide Web and the Perl programming
language.

--------------------------------------------------------------------------------

GG Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

OTHER SUPPORT
(Use continuation pages if necessary)

FOLLOW INSTRUCTIONS CAREFULLY. Incomplete, inaccurate, or ambiguous information
about OTHER SUPPORT could lead to significant delays in the review and/or
possible funding of the application. If there are changes in the information
after submission, notify the scientific review administrator or the initial
review group before the review; if changes occur after the review, notify the
appropriate institute.

Other support is defined as all funds or resources, whenever Federal,
non-Federal, or institutional, available to the principal investigator/program
director (and other key personnel named in the application) in direct support of
their research endeavors through research or training grants, cooperative
agreements, contracts fellowships, gifts, prizes, and other means. Key personnel
are defined as all individuals who participate in the scientific development or
execution of the project. Key personnel typically will include all individuals
with doctoral or other professional degrees, but in some projects will include
individuals at the masters or baccalaureate level provided they contribute in a
substantive way to the scientific development or execution of the project.

Name Gregory T. Went. Ph.D Active X Pending None
----------------------------------- ------ ------- --------

a. Source and identifying no. NIST 70NANB5H1036 P.I. Gregory T. Went
---------------------- -----------------------

Title Integrated Microfabricated DNA Analysis Device for Diagnosis of Complex
-------------------------------------------------------------------------
Genetic Disorder
----------------

b. Your role on project Principal Investigator [XXXXXXXXXXXXXXXX]
---------------------------- -------------------

c. Dates and costs of entire project [XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]
-------------------------------------------

d. Dates and costs of current year [XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]
---------------------------------------------

e. Specific aims of project This project proposes to develop a device to
----------------------------------------------------
extend the use of DNA analysis from the characterization of simple inborn
--------------------------------------------------------------------------------
genetic disorders to the efficient diagnosis and treatment of complex illnesses
--------------------------------------------------------------------------------
like cardiovascular disease and cancer.
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

f. Describe scientific and budgetary overlap This present application contains
-----------------------------------
optical components similar to those described herein.
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

g. Describe adjustments you will make if the present application is funded
(budget, % effort, aims, etc.)

Adjustments will be made to the aims of the NIST project during 1997 if an award
--------------------------------------------------------------------------------
is received.
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

[Confidential Treatment Requested]

GG Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

OTHER SUPPORT
(Use continuation pages if necessary)

Other support is defined as all funds or resources, whether Federal, non-
Federal, or institutional, available to the principal investigator/program
director (and other key personnel named in the application) in direct support of
their research endeavors through research or training grants, cooperative
agreements, contracts, fellowships, gifts, prizes, and other means. Key
personnel are defined as all individuals who participate in the scientific
development or execution of the project. Key personnel typically will include
all individuals with doctoral or other professional degrees, but in some
projects will include individuals at the masters or baccalaureate level provided
they contribute in a substantive way to the scientific development or execution
of the project.

Name Gregory T. Went, Ph.D Active X Pending None
----------------------------------- ------ ------- --------

a. Source and identifying no. NIST 70NANB5H1066 P.I. Jonathon M. Rothberg
---------------------- -----------------------

Title Molecular Recognition Technology for Precise Design of Protein-Specific
-------------------------------------------------------------------------
Drugs
-----

b. Your role on project Business Administrator [XXXXXXXXXXXXX]
---------------------------- -------------------

c. Dates and costs of entire project [XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]
-------------------------------------------

d. Dates and costs of current year [XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]
---------------------------------------------

e. Specific aims of project This project, through state-of-the-art molecular
----------------------------------------------------
biology, structural physics, and computation methods, proposes to understand how
--------------------------------------------------------------------------------
small highly constrained mimics of protein modules bind to a target protein.
--------------------------------------------------------------------------------
This technological discovery uncovers rules governing protein recognition which
--------------------------------------------------------------------------------
can be applied to guide the development of drugs to combat major uncured
--------------------------------------------------------------------------------
diseases.
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

f. Describe scientific and budgetary overlap No Scientific or budgetary
-----------------------------------
overlap
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

g. Describe adjustments you will make if the present application is funded
(budget, % effort, aims, etc.)

Dr. Went's effort will be reduced to [XXX] if the current project is funded.
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

[Confidential Treatment Requested]

GG Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

OTHER SUPPORT
(Use continuation pages if necessary)

Other support is defined as all funds or resources, whether Federal, non-
Federal, or institutional, available to the principal investigator/program
director (and other key personnel named in the application) in direct support of
their research endeavors through research or training grants, cooperative
agreements, contracts fellowships, gifts, prizes, and other means. Key personnel
are defined as all individuals who participate in the scientific development or
execution of the project. Key personnel typically will include all individuals
with doctoral or other professional degrees, but in some projects will include
individuals at the masters or baccalaureate level provided they contribute in a
substantive way to the scientific development or execution of the project.

Name Gregory T. Went, Ph.D Active X Pending None
----------------------------------- ------ ------- --------

a. Source and identifying no. NIH SBIR 2 R44 HGOO960-02 P.I. Gregory T. Went
---------------------------- -----------------

Title Modular UltraHigh ThroughPut DNA Sequencing
-------------------------------------------------------------------------

b. Your role on project Principal Investigator [XXXXXXXXXXX]
---------------------------- -------------------

c. Dates and costs of entire project [XXXXXXXXXXXXXXXXXXXXXXXXXXXX]
-------------------------------------------

d. Dates and costs of current year [XXXXXXXXXXXXXXXXXXXXXXXXXXXX]
---------------------------------------------

e. Specific aims of project This project proposes to develop a working
----------------------------------------------------
prototype incorporating a microfabricated separation module and loading
--------------------------------------------------------------------------------
interface.
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

f. Describe scientific and budgetary overlap This project serves as the basis
-----------------------------------
to the microchannel structures needed for the current proposal.
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

g. Describe adjustments you will make if the present application is funded
(budget, % effort, aims, etc.)

No Adjustments will be necessary
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

[Confidential Treatment Requested]

GG Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

OTHER SUPPORT
(Use continuation pages if necessary)

Name Jonathon M. Rothberg, Ph.D Active X Pending None
----------------------------------- ------ ------- --------

a. Source and identifying no. NIST 70NANB5H1036 P.I. Gregory T. Went
---------------------- -----------------------

Title Integrated Microfabricated DNA Analysis Device for Diagnosis of Complex
-------------------------------------------------------------------------
Genetic Disorder
----------------

b. Your role on project Business Administrator [XXXXXXXXXXX]
---------------------------- -------------------

c. Dates and costs of entire project [XXXXXXXXXXXXXXXXXXXXXXXXXXXXX]
-------------------------------------------

d. Dates and costs of current year [XXXXXXXXXXXXXXXXXXXXXXXXXXXXX]
---------------------------------------------

--------------------------------------------------------------------------------

g. Describe adjustments you will make if the present application is funded
(budget, % effort, aims, etc.)

--------------------------------------------------------------------------------

[Confidential Treatment Requested]

GG Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

OTHER SUPPORT
(Use continuation pages if necessary)

Name Jonathon M. Rothberg, Ph.D Active X Pending None
----------------------------------- ------ ------- --------

a. Source and identifying no. NIST 70NANB5H1066 P.I. Jonathon M. Rothberg
---------------------- -----------------------

Title Molecular Recognition Technology for Precise Design of Protein-Specific
-------------------------------------------------------------------------
Drugs
-------------------------------------------------------------------------

b. Your role on project Principal Investigator [XXXXXXXXXXXXXX]
---------------------------- -------------------

c. Dates and costs of entire project [XXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]
-------------------------------------------

d. Dates and costs of current year [XXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]
---------------------------------------------

--------------------------------------------------------------------------------

f. Describe scientific and budgetary overlap No Scientific or budgetary
-----------------------------------
overlap
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

g. Describe adjustments you will make if the present application is funded
(budget, % effort, aims, etc.)

Dr. Rothberg's effort will be reduced to [XXX] if the current project is funded.
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

[Confidential Treatment Requested]

GG Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

OTHER SUPPORT
(Use continuation pages if necessary)

Other support is defined as all funds or resources, whether Federal,
non-Federal, or institutional, available to the principal investigator/program
director (and other key personnel named in the application) in direct support of
their research endeavors through research or training grants, cooperative
agreements, contracts fellowships, gifts, prizes, and other means. Key personnel
are defined as all individuals who participate in the scientific development or
execution of the project. Key personnel typically will include all individuals
with doctoral or other professional degrees, but in some projects will include
individuals at the masters or baccalaureate level provided they contribute in a
substantive way to the scientific development or execution of the project.

Name Jonathon M. Rothberg, Ph.D Active X Pending None
----------------------------------- ------ ------- --------

a. Source and identifying no. NIH SBIR 2 R44 HG00960-02 P.I. Gregory T. Went
---------------------------- -----------------

Title Modular UltraHigh Throughput DNA Sequencing
-------------------------------------------------------------------------

b. Your role on project Investigator [XXXXXXXXXXX]
---------------------------- -------------------

c. Dates and costs of entire project [XXXXXXXXXXXXXXXXXXXXXXXXXX]
-------------------------------------------

d. Dates and costs of current year [XXXXXXXXXXXXXXXXXXXXXXXXXXX]
---------------------------------------------

--------------------------------------------------------------------------------

f. Describe scientific and budgetary overlap This project serves as the basis
-----------------------------------
to the michrochannel structures needed for the current proposal.
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

g. Describe adjustments you will make if the present application is funded
(budget, % effort, aims, etc.)

No Adjustments will be necessary
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------
55

[Confidential Treatment Requested]

GG Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

OTHER SUPPORT
(Use continuation pages if necessary)

Other support is defined as all funds or resources, whether Federal,
non-Federal, or institutional, available to the principal investigator/program
director (and other key personnel named in the application) in direct support of
their research endeavors through research or training grants, cooperative
agreements, contracts fellowships, gifts, prizes, and other means. Key personnel
are defined as all individuals who participate in the scientific development or
execution of the project. Key personnel typically will include all individuals
with doctoral or other professional degrees, but in some projects will include
individuals at the masters or baccalaureate level provided they contribute in a
substantive way to the scientific development or execution of the project.

Name Michael P. McKenna, Ph.D Active X Pending None
----------------------------------- ------ ------- --------

a. Source and identifying no. NIST 70NANB5H1036 P.I. Gregory T. Went
---------------------- -----------------------

Title Integrated Microfabricated DNA Analysis Device for Diagnosis of Complex
-------------------------------------------------------------------------
Genetic Disorder
-------------------------------------------------------------------------

b. Your role on project Senior Research Scientist [XXXXXXXXXXX]
---------------------------- -------------------

c. Dates and costs of entire project [XXXXXXXXXXXXXXXXXXXXXXXXXXX]
-------------------------------------------

d. Dates and costs of current year [XXXXXXXXXXXXXXXXXXXXXXXXXXXX]
---------------------------------------------

--------------------------------------------------------------------------------

g. Describe adjustments you will make if the present application is funded
(budget, % effort, aims, etc.)

Adjustments will be made to the aims of the NIST during 1997 if an award is
--------------------------------------------------------------------------------
received.
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------
56

[Confidential Treatment Requested]

GG Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

OTHER SUPPORT
(Use continuation pages if necessary)

Name John W. Simpson Active X Pending None
----------------------------------- ------ ------- --------

a. Source and identifying no. NIST 70NANB5H1036 P.I. Gregory T. Went
---------------------- -----------------------

b. Your role on project Senior Research Engineer [XXXXXXXXXX]
---------------------------- -------------------

c. Dates and costs of entire project [XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]
-------------------------------------------

d. Dates and costs of current year [XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]
---------------------------------------------

--------------------------------------------------------------------------------

g. Describe adjustments you will make if the present application is funded
(budget, % effort, aims, etc.)

--------------------------------------------------------------------------------

[Confidential Treatment Requested]

GG Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

OTHER SUPPORT
(Use continuation pages if necessary)

Name John W. Simpson Active X Pending None
----------------------------------- ------ ------- --------

a. Source and identifying no. NIST SBIR 2 R44 HG00960-02 P.I. Gregory T. Went
---------------------------- -----------------

Title Modular UltraHigh Throughput DNA Sequencing
-------------------------------------------------------------------------

b. Your role on project Senior Engineer [XXXXXXXXXXXX]
---------------------------- -------------------

c. Dates and costs of entire project [XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]
-------------------------------------------

d. Dates and costs of current year [XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]
---------------------------------------------

--------------------------------------------------------------------------------

g. Describe adjustments you will make if the present application is funded
(budget, % effort, aims, etc.)

No Adjustments will be necessary
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------
58

[Confidential Treatment Requested]

GG Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

OTHER SUPPORT
(Use continuation pages if necessary)

Name Harold G. Craighead Active X Pending None
----------------------------------- ------ ------- --------

a. Source and identifying no. ARPA/AF - F49620-93-C-0061 P.I. Craighead
---------------------------- ----------------

Title Micro-Column Arrays for Nanolithography
-------------------------------------------------------------------------

b. Your role on project % Effort
---------------------------- -------------------

c. Dates and costs of entire project [XXXXXXXXXXXXXXXXXXXXXXXXXXXX]
-------------------------------------------

d. Dates and costs of current year [XXXXXXXXXXXXXXXXXXXXXXXXXXXXX]
---------------------------------------------

e. Specific aims of project Develop a miniaturized electron beam source and
----------------------------------------------------
optical column
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

f. Describe scientific and budgetary overlap None
-----------------------------------

--------------------------------------------------------------------------------

g. Describe adjustments you will make if the present application is funded
(budget, % effort, aims, etc.)

None
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------
59

[Confidential Treatment Requested]

GG Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
================================================================================
OTHER SUPPORT
(Use continuation pages if necessary)

FOLLOW INSTRUCTIONS CAREFULLY. Incomplete, inaccurate, or ambiguous information
about OTHER SUPPORT could lead to significant delays in the review and/or
possible funding of the application. If there are changes in the information
after submission, notify the scientific review administrator of the initial
review group before the review; if changes occur after the review, notify the
appropriate insurance.

Other support is defined as all funds or resources, whether Federal,
non-Federal, or institutional, available to the principal investigator/program
(director and other key personnel named in the application) in direct support of
their research endeavors through research or training grants, cooperative
agreements, contracts, fellowships, gifts, prizes, and other means. Key
personnel are defined as all individuals who participate in the scientific
development or execution of the project. Key personnel typically will include
all individuals with doctoral or other professional degrees, but in some
projects will include individuals at the masters or baccalaureate level provided
they contribute in a substantive way to the scientific development or execution
of the project.

Reporting requirements are: for each of the key personnel, describe (1) all
currently active support and (2) all applications and proposals pending review
or award, whether related to the application or not. If the support is part of
a larger project, identify the principal investigator/program director and
provide the data for the relevant subproject(s). If an individual has no active
or pending support, check "None." Use continuation pages as needed to provide
the required information in the format as shown below.

Name Harold G. Craighead Active X Pending None
-------------------------------- ------ ------ ------

a. Source and identifying no. ARPA/ONR - NOOO14-93-1-1080 P.I. Craighead
-------------------------------- -------------

Title Molecular Approaches to Device Nanofabrication
---------------------------------------------------------------------------

b. Your role on project PI % Effort
---------- -------------------- ---------------

c. Dates and costs of entire project [XXXXXXXXXXXXXXXXXXXXXXXXXXX]
-------------------------------------------

d. Dates and costs of current year [XXXXXXXXXXXXXXXXXXXXXXXXXXXX]
---------------------------------------------

e. Specific aims of project Investigate the properties and use of
----------------------------------------------------
self-assembled organic monolayers as resists for nanofabrication
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

f. Describe scientific and budgetary overlap None
-----------------------------------

--------------------------------------------------------------------------------

g. Describe adjustments you will make if the present application is funded
(budget, % effort, aims, etc.)

None
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

================================================================================

[Confidential Treatment Requested]

GG Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

OTHER SUPPORT
(Use continuation pages if necessary)

Name Harold G. Craighead Active X Pending None
----------------------------------- ------ ------- --------

a. Source and identifying no. DoD/Rome Labs - F30602-93-C-0061
--------------------------------------------------

P.I. Craighead
----------------------------------------------------------------------------

Title Advancement of Free-Space Optical Interconnects
-------------------------------------------------------------------------

b. Your role on project PI [XXXXXXXXXXXXXXXXX] Summer
-------- -------- --------- ---------

c. Dates and costs of entire project [XXXXXXXXXXXXXXXXXXXXXXXXXXXX]
-------------------------------------------

d. Dates and costs of current year [XXXXXXXXXXXXXXXXXXXXXXXXXXXX]
---------------------------------------------

e. Specific aims of project Fabricate and test an optical interconnect for
----------------------------------------------------
electronic circuits based on microfabricated diffractive optics.
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

f. Describe scientific and budgetary overlap Uses advanced microfabrication
-----------------------------------
technologies
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

g. Describe adjustments you will make if the present application is funded
(budget, % effort, aims, etc.)

None
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

[Confidential Treatment Requested]

Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

OTHER SUPPORT
(Use continuation pages if necessary)

Name Harold G. Craighead Active X Pending None
----------------------------------- ------ ------- --------

a. Source and identifying no. MSC/NSF - DMR-9121654 P.I. Craighead
--------------------------- -------------------

Title Electron Scattering in Mesoscopic Systems
-------------------------------------------------------------------------

b. Your role on project PI % Effort
---------- -------------- -------------------

c. Dates and costs of entire project
-------------------------------------------

d. Dates and costs of current year [XXXXXXXXXXXXXXXXXXXXXXXXXXXXX]
---------------------------------------------

e. Specific aims of project Explore electron scattering in small electronic
----------------------------------------------------
devices
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

f. Describe scientific and budgetary overlap None
-----------------------------------

--------------------------------------------------------------------------------

g. Describe adjustments you will make if the present application is funded
(budget, % effort, aims, etc.)

None
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

[Confidential Treatment Requested]

Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

OTHER SUPPORT
(Use continuation pages if necessary)

Name Harold G. Craighead Active X Pending None
----------------------------------- ------ ------- --------

a. Source and identifying no. Cornell Center for Advanced Tech
--------------------------------------------------

P.I. Craighead
----------------------------------------------------------------------------

Title Nanofabrication of Artificial Insect Eggs for use in Biological Control
-------------------------------------------------------------------------
of Parasites
-------------------------------------------------------------------------

b. Your role on project PI % Effort
----------- ------------- -------------------

c. Dates and costs of entire project (For renewals, include only the most
recent competitive award. List direct and indirect costs separately.)

[XXXXXXXXXXXXXXXXXXXXXXXXXXXX]
--------------------------------------------------------------------------------

d. Dates and costs of current year
---------------------------------------------

e. Specific aims of project This study involves creating micro-structures
----------------------------------------------------
on the surface of artificial insect eggs to encourage egg laying by beneficial
--------------------------------------------------------------------------------
insect parasites. The goal is to mass rear beneficial parasites in artificial
--------------------------------------------------------------------------------
eggs.
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

f. Describe scientific and budgetary overlap None
-----------------------------------

--------------------------------------------------------------------------------

g. Describe adjustments you will make if the present application is funded
(budget, % effort, aims, etc.)

None
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

[Confidential Treatment Requested]

Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
-----------------
--------------------------------------------------------------------------------

OTHER SUPPORT
(Use continuation pages if necessary)

Other support is defined as all funds or resources, whether Federal, non-
Federal, or institutional, available to the principal investigator/program
director (and other key personnel named in the application) in direct support of
their research endeavors through research or training grants, cooperative
agreements, contracts, fellowships, gifts, prizes, and other means. However, in
the case of prizes and gifts, only those that support the specific project must
be reported. Key personnel are defined as all individuals who participate in the
scientific development or execution of the project. Key personnel typically will
include all individuals with doctoral or other professional degrees, but in some
projects will include individuals at the masters or baccalaureate level provided
they contribute in a substantive way to the scientific development or execution
of the project.

Reporting requirements are: for each of the key personnel, describe (1) all
currently active support and (2) all applications and proposals pending review
or award whether related to this application or not. If the support is part of a
larger project, identify the principal investigator/program director and provide
the data for the relevant subproject(s). If an individual has no active or
pending support, check "None." Use continuation pages as needed to provide the
required information in the format as shown below. Information may be combined
as long as the format remains the same. For example, all key personnel who have
no other support may be listed on a single page. DO NOT SEND in a separate page
for each person listed for whom "None" is checked.

Name Harold G. Craighead Active Pending X None
----------------------------------- ------ ------- --------

a. Source and identifying no. NY State Health Dept/NIH P.I. William Shain
---- --- ----------------

Title Brain Prostheses: Tissue Compatibility and Integration
------ ---------------

b. Your role on project PI of Subcontract from Cornell % Effort
--- ------ --------

c. Dates and costs of entire project (For renewals, include only the most
recent competitive award. List direct and indirect costs separately.)

[XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]
--------------------------------------------------------------------------------

d. Dates and costs of current year
---------------------------------------------

e. Specific aims of project Develop surface coatings and treatments for the
----------------------------------------------------
selective attachment of neurons to electrodes
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

f. Describe scientific and budgetary overlap None
-----------------------------------

--------------------------------------------------------------------------------

g. Describe adjustments you will make if the present application is funded
(budget, % effort, aims, etc.)

None
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

[Confidential Treatment Requested]

GG Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

OTHER SUPPORT
(Use continuation pages if necessary)

Other support is defined as all funds or resources, whether Federal, non-
Federal, or institutional, available to the principal investigator/program
director (and other key personnel named in the application) in direct support of
their research endeavors through research or training grants, cooperative
agreements, contracts, fellowships, gifts, prizes, and other means. However, in
the case of prizes and gifts, only those that support the specific project must
be reported. Key personnel are defined as all individuals who participate in the
scientific development or execution of the project. Key personnel typically will
include all individuals with doctoral or other professional degrees, but in some
projects will include individuals at the masters or baccalaureate level provided
they contribute in a substantive way to the scientific development or execution
of the project.

Name Harold G. Craighead Active X Pending None
----------------------------------- ------ ------- --------

a. Source and identifying no. CuraGen Corp. P.I. Craighead
---------------------- -----------------------

Title Nanofabrication of Devices for Genetic Analysis
-------------------------------------------------------------------------

b. Your role on project PI % Effort
---------------------------- -------------------

c. Dates and costs of entire project (For renewals, include only the most
recent competitive award. List direct and indirect costs separately.)

[XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]
--------------------------------------------------------------------------------

d. Dates and costs of current year
---------------------------------------------

e. Specific aims of project This project is to find efficient ways of
----------------------------------------------------
microfabricating multiple parallel lanes for conventional gel electrophoresis.
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

f. Describe scientific and budgetary overlap Gel channels made under this
-----------------------------------
project can be used with the proposed optics.
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

g. Describe adjustments you will make if the present application is funded
(budget, % effort, aims, etc.)

None
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

[Confidential Treatment Requested]

Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
-------------------------------------------------------------------------------

OTHER SUPPORT
(Use continuation pages if necessary)

Other support is defined as all funds or resources, whether Federal, non-
Federal, or institutional, available to the principal investigator/program
director (and other key personnel named in the application) in direct support of
their research endeavors through research or training grants, cooperative
agreements, contracts, fellowships, gifts, prizes, and other means. However, in
the case of prizes and gifts, only those that support the specific project must
be reported.Key personnel are defined as all individuals who participate in the
scientific development or execution of the project. Key personnel typically will
include all individuals with doctoral or other professional degrees, but in some
projects will include individuals at the masters or baccalaureate level provided
they contribute in a substantive way to the scientific development or execution
of the project.

Name Harold G. Craighead Active X Pending None
----------------------------------- ------ ------- --------

a. Source and identifying no. DOD/MPO P.I. Craighead
---------------------- -----------------------

Title Electron Beam Programming
-------------------------------------------------------------------------

b. Your role on project PI % Effort
---------- ----------------- -------------------

c. Dates and costs of entire project (For renewals, include only the most
recent competitive award. List direct and indirect costs separately.)

[XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]
--------------------------------------------------------------------------------

d. Dates and costs of current year
---------------------------------------------

e. Specific aims of project Electron beam programming of MOS transistor
----------------------------------------------------
memories
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

f. Describe scientific and budgetary overlap None
-----------------------------------

--------------------------------------------------------------------------------

g. Describe adjustments you will make if the present application is funded
(budget, % effort, aims, etc.)

None
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

[Confidential Treatment Requested]

Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
-------------------------------------------------------------------------------

OTHER SUPPORT
(Use continuation pages if necessary)

Other support is defined as all funds or resources, whether Federal, non-
Federal, or institutional, available to the principal investigator/program
director (and other key personnel named in the application) in direct support of
their research endeavors through research or training grants, cooperative
agreements, contracts, fellowships, gifts, prizes, and other means. However, in
the case of prizes and gifts, only those that support the specific project must
be reported.Key personnel are defined as all individuals who participate in the
scientific development or execution of the project. Key personnel typically will
include all individuals with doctoral or other professional degrees, but in some
projects will include individuals at the masters or baccalaureate level provided
they contribute in a substantive way to the scientific development or execution
of the project.

Name Herbert H. Hooper Active X Pending None
----------------------------------- ------ ------- --------

a. Source and identifying no. NIH HG01289-01 P.I. H. Hooper
---------------------- -----------------------

Title Replaceable Gels for Microchannel DNA Electrophoresis
-------------------------------------------------------------------------

b. Your role on project PI [XXXXXXXXXXX]
-------------------------- -------------------

c. Dates and costs of entire project (For renewals, include only the most
recent competitive award. List direct and indirect costs separately.)

[XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]
--------------------------------------------------------------------------------

d. Dates and costs of current year same
---------------------------------------------

e. Specific aims of project Develop replaceable matrices for DNA sequencing
----------------------------------------------------
and typing by capillary electrophoresis with low viscosity during loading and
--------------------------------------------------------------------------------
unloading.
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

f. Describe scientific and budgetary overlap None. Related area but different
-----------------------------------
approach and technology.
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

g. Describe adjustments you will make if the present application is funded
(budget, % effort, aims, etc.)

None
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

[Confidential Treatment Requested]

Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

OTHER SUPPORT
(Use continuation pages if necessary)

Other support is defined as all funds or resources, whether Federal, non-
Federal, or institutional, available to the principal investigator/program
director (and other key personnel named in the application) in direct support of
their research endeavors through research or training grants, cooperative
agreements, contracts, fellowships, gifts, prizes, and other means. However, in
the case of prizes and gifts, only those that support the specific project must
be reported. Key personnel are defined as all individuals who participate in the
scientific development or execution of the project. Key personnel typically will
include all individuals with doctoral or other professional degrees, but in some
projects will include individuals at the masters or baccalaureate level provided
they contribute in a substantive way to the scientific development or execution
of the project.

Name Herbert H. Hooper Active X Pending None
----------------------------------- ------ ------- --------

H. Hooper (Soane)
a. Source and identifying no. NIST 70NANB5H1036 P.I. G. Went (CuraGen)
---------------------- -----------------------

Title Integrated Microfabricated DNA Analysis Device
-------------------------------------------------------------------------

b. Your role on project PI at Soane [XXXXXXXXXXXXXXXX]
---------------------------- -------------------

c. Dates and costs of entire project (For renewals, include only the most
recent competitive award. List direct and indirect costs separately.)

[XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]
--------------------------------------------------------------------------------

d. Dates and costs of current year [XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
---------------------------------------------
XXXXXXXXXXXXXXXXXXXXXXXXXXXX]
--------------------------------------------------------------------------------

e. Specific aims of project Develop replaceable gels for microfabricated
----------------------------------------------------
device that flow at room temperature and gel upon heating.
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

g. Describe adjustments you will make if the present application is funded
(budget, % effort, aims, etc.)

None
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

[Confidential Treatment Requested]

GG Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

OTHER SUPPORT
(Use continuation pages if necessary)

Other support is defined as all funds or resources, whenever Federal, non-
Federal, or institutional, available to the principal investigator/program
director (and other key personnel named in the application) in direct support of
their research endeavors through research or training grants, cooperative
agreements, contracts fellowships, gifts, prizes, and other means. However, in
the case of prizes and gifts, only those that support the specific project must
be reported. Key personnel are defined as all individuals who participate in the
scientific development or execution of the project. Key personnel typically will
include all individuals with doctoral or other professional degrees, but in some
projects will include individuals at the masters or baccalaureate level provided
they contribute in a substantive way to the scientific development or execution
of the project.

Name Herbert H. Hooper Active X Pending None
----------------------------------- ------ ------- --------

a. Source and identifying no. NIH HGO1338-01 P.I. A.P. Sassi
---------------------- -----------------------

Title Constant - Field Electrophoresis of Large DNA
-------------------------------------------------------------------------

b. Your role on project Scientist [XXXXXXXXXXX]
---------------------------- -------------------

c. Dates and costs of entire project (For renewals, include only the most
recent competitive award. List direct and indirect costs separately.)

[XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]
--------------------------------------------------------------------------------

d. Dates and costs of current year [XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
---------------------------------------------
XXXXXXXXXXXXXXXXXXXXXXXXX]
--------------------------------------------------------------------------------

e. Specific aims of project Develop gels for separates large (greater than
----------------------------------------------------
50kbp) DNA fragments without pulsed fields.
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

f. Describe scientific and budgetary overlap None
-----------------------------------

--------------------------------------------------------------------------------

g. Describe adjustments you will make if the present application is funded
(budget, % effort, aims, etc.)

None
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

[Confidential Treatment Requested]

GG Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

OTHER SUPPORT
(Use continuation pages if necessary)

Other support is defined as all funds or resources, whether Federal, non-
Federal, or institutional, available to the principal investigator/program
director (and other key personnel named in the application) in direct support of
their research endeavors through research or training grants, cooperative
agreements, contracts, fellowships, gifts, prizes, and other means. However, in
the case of prizes and gifts, only those that support the specific project must
be reported. Key personnel are defined as all individuals who participate in the
scientific development or execution of the project. Key personnel typically will
include all individuals with doctoral or other professional degrees, but in some
projects will include individuals at the masters or baccalaureate level provided
they contribute in a substantive way to the scientific development or execution
of the project.

Name David Soane Active X Pending None
----------------------------------- ------ ------- --------

a. Source and identifying no. NIH HGO1338-01 P.I. A.P. Sassi
---------------------- -----------------------

Title Constant - Field Electrophoresis of Large DNA
-------------------------------------------------------------------------

b. Your role on project Scientist [XXXXXXXXXXXXX]
---------------------------- -------------------

c. Dates and costs of entire project (For renewals, include only the most
recent competitive award. List direct and indirect costs separately.)

[XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]
--------------------------------------------------------------------------------

[XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX] [XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
---------------------------------------------
XXXXXXXXXXXXXXXXXXXXXXXXX]
--------------------------------------------------------------------------------

e. Specific aims of project Develop gels for separating large (greater than
----------------------------------------------------
50kbp) DNA fragments without pulsed fields
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

f. Describe scientific and budgetary overlap None
-----------------------------------

--------------------------------------------------------------------------------

g. Describe adjustments you will make if the present application is funded
(budget, % effort, aims, etc.)

None
--------------------------------------------------------------------------------

[Confidential Treatment Requested]

GG Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

OTHER SUPPORT
(Use continuation pages if necessary)

Other support is defined as all funds or resources, whenever Federal, non-
Federal, or institutional, available to the principal investigator/program
director (and other key personnel named in the application) in direct support of
their research endeavors through research or training grants, cooperative
agreements, contracts fellowships, gifts, prizes, and other means. However, in
the case of prizes and gifts, only those that support the specific project must
be reported. Key personnel are defined as all individuals who participate in the
scientific development or execution of the project. Key personnel typically will
include all individuals with doctoral or other professional degrees, but in some
projects will include individuals at the masters or baccalaureate level provided
they contribute in a substantive way to the scientific development or execution
of the project.

Name David Soane Active X Pending None
----------------------------------- ------ ------- --------

a. Source and identifying no. NIH HG01289-01 P.I. H. Hooper
---------------------- -----------------------

Title Replaceable Gels for Microchannel DNA Electrophoresis
-------------------------------------------------------------------------

b. Your role on project Scientist [XXXXXXXXXX]
---------------------------- -------------------

c. Dates and costs of entire project (For renewals, include only the most
recent competitive award. List direct and indirect costs separately.)

[XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]
--------------------------------------------------------------------------------

d. Dates and costs of current year [XXXX]
---------------------------------------------

--------------------------------------------------------------------------------

g. Describe adjustments you will make if the present application is funded
(budget, % effort, aims, etc.)

None.
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

[Confidential Treatment Requested]

GG Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

OTHER SUPPORT
(Use continuation pages if necessary)

Other support is defined as all funds or resources, whenever Federal, non-
Federal, or institutional, available to the principal investigator/program
director (and other key personnel named in the application) in direct support of
their research endeavors through research or training grants, cooperative
agreements, contracts fellowships, gifts, prizes, and other means. However, in
the case of prizes and gifts, only those that support the specific project must
be reported. Key personnel are defined as all individuals who participate in the
scientific development or execution of the project. Key personnel typically will
include all individuals with doctoral or other professional degrees, but in some
projects will include individuals at the masters or baccalaureate level provided
they contribute in a substantive way to the scientific development or execution
of the project.

Name Alexander P. Sassi Active X Pending None
----------------------------------- ------ ------- --------

a. Source and identifying no. NIH HG01338-01 P.I. A.P. Sassi
---------------------- -----------------------

Title Constant - Field Electrophoresis of Large DNA
-------------------------------------------------------------------------

b. Your role on project Scientist [XXXXXXXX]
---------------------------- -------------------

c. Dates and costs of entire project (For renewals, include only the most
recent competitive award. List direct and indirect costs separately.)

[XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]
--------------------------------------------------------------------------------

[XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX] [XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
---------------------------------------------
XXXXXXXXXXXXXXXXXXXXX]
--------------------------------------------------------------------------------

e. Specific aims of project Develop gels for separating large (greater than
----------------------------------------------------
50kbp) DNA fragments without pulsed fields.
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

f. Describe scientific and budgetary overlap None. Project ends before subject
-----------------------------------
grant will begin.
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

g. Describe adjustments you will make if the present application is funded
(budget, % effort, aims, etc.)

None.
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

[Confidential Treatment Requested]

GG Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

OTHER SUPPORT
(Use continuation pages if necessary)

Name Trevor Hawkins Active X Pending None
----------------------------------- ------ ------- --------

a. Source and identifying no. Department of Energy DE-FGO2-95ER62099
--------------------------------------------------

P.I. Trevor L. Hawkins
-----------------------

Title DNA Sample Munipulation and Automation
-------------------------------------------------------------------------

b. Your role on project Principal Investigator [XXXXXXXXX]
---------------------------- -------------------

c. Dates and costs of entire project [XXXXXXXXXXXXXXXXXXXXXXXXXXX]
-------------------------------------------

d. Dates and costs of current year [XXXXXXXXXXXXXXXXXXXXXXXXXXX]
---------------------------------------------

e. Specific aims of project The Aims of this Project are:
----------------------------------------------------
1) Produce sub-clone libraries from a variety of templates and automate
--------------------------------------------------------------------------------
library construction process.
--------------------------------------------------------------------------------
2) Sequence large numbers of DNA templates for analysis on sequencers and
--------------------------------------------------------------------------------
capillary systems.
--------------------------------------------------------------------------------
3) Evaluate capillary array for DNA sequence while integrating and automating
--------------------------------------------------------------------------------
the procedures from MAP clone to get ready samples.
--------------------------------------------------------------------------------

f. Describe scientific and budgetary overlap No Scientific or budgetary
-----------------------------------
overlap
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

g. Describe adjustments you will make if the present application is funded
(budget, % effort, aims, etc.)

No adjustment is necessary.
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

[Confidential Treatment Requested]

GG Principal Investigator/Program Director (Last, first, middle)
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

Name Trevor L. Hawkins Active Pending X None
--------------------------------- --------- ----- -------

a. Source and identifying no. NIH RFA HG95-001 with Intelligent Automation
---------------------------------------------------
P.I. Steve Gordon
---------------------------------

Title Improved Electrophoretic DNA Sequencing Technology
---------------------------------------------------------------------------

b. Your role on project: Co-Investigator [XXXXXXXXXXX]
--------------------------------- ---------------

c. Dates and costs of entire project: Dates and Costs Currently Under Review
-------------------------------------------

d. Date and costs of current year: Dates and Costs Currently Under Review
----------------------------------------------

e. Specific aims of project: The Aims of this Project are: Studies using gel
----------------------------------------------------
electrophoresis systems that could yield reductions in scale and increased
--------------------------------------------------------------------------------
throughput of DNA sequencing.
--------------------------------------------------------------------------------

f. Describe scientific and buggetary overlap: Effort will be reduced if there is
-----------------------------------
overlap.
--------------------------------------------------------------------------------

f. Describe adjustments you will make if the present application is funded
(budget % effort, aims, etc.)

Effort will be reduced if RFA HG 95-004 is funded.
--------------------------------------------------------------------------------

OTHER SUPPORT CONTINUED:

NAME ACTIVE PENDING
-----------------------------------------------------

Lincoln Stein None None
William Lee None None

--------------------------------------------------------------------------------

[Confidential Treatment Requested]

HH Principal Investigator/Program Director (Last, first, middle)
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

RESOURCES AND ENVIRONMENT
(CuraGen Corporation)
--------------------------------------------------------------------------------

FACILITIES. Mark the facilities to be used at each performance site listed in
item 9. Face Page, and briefly indicate their capabilities, relative
capabilities, relative proximity, and extent of availability to the project. Use
"Other" to describe the facilities at any other performance sites listed in item
9 on the Face Page and at sites for field studies. Use continuation pages if
necessary. Include an explanation of any consortium/contractual arrangements
with other organizations.

[X] Laboratory: CuraGen has 8000 sq. ft. of state-of-the-art molecular and
structural biology laboratory and office space at its research headquarters in
Branford. A recent buildout of 3000 square feet of space was completed for
structural biology and computation laboratories. This space also houses the
Company's 1000 sequence/day DNA analysis facility and 3500 square feet of
molecular biology facilities, which includes standard equipment. By 1996, the
Company plans to occupy over 15,000 sq. ft. of lab and office space.

[_] Clinical:

[_] Animal:

[X] Computer: Four workstation class SGI computers are available for CuraGen's
software development. Twenty Macintoshes and PowerPC's are also onsite. Large
scale sequence databases, such as GenBank, are available on-site, as are
literature search databases such as CAS on-line. Entrez and MedLine.

[X] Office: Ample (2500 sq. Ft.) office space (with a library in our new
expansion) is available for CuraGen's 25 employees.

[X] Other ( ): CuraGen collaborates closely with all three coinvestigators as a
part of ongoing projects. In addition, CuraGen has a visiting scientist position
at MIT/The Whitehead Institute with office and computer access.

--------------------------------------------------------------------------------
MAJOR EQUIPMENT List the most important equipment items already available for
this project, noting the location and pertinent capabilities or each

CuraGen has developed under NIH and NIST funding a high throughput DNA analysis
instrument, placing in its core facility. These systems are driven by TECAN and
Beckman robot stations with integrated thermal cycling (384 well). Our molecular
biology facilities are modern and well equipped with freezers, incubators, PCR,
HPLC, electroporation, electrophoresis, centrifuges, plate readers, microscopes
and hoods.

--------------------------------------------------------------------------------
ADDITIONAL INFORMATION: Provide any other information describing the environment
for the project. Identify support services such as consultant, secretarial,
machine shop, and electronics shop, and the extent to which they will be
available to the project.

Secretarial, administrative support, and accounting are all provided onsite, as
are reporting and regulatory support.

--------------------------------------------------------------------------------

HH Principal Investigator/Program Director (Last, first, middle)
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

RESOURCES AND ENVIRONMENT

(Cornell University)
--------------------------------------------------------------------------------

FACILITIES Mark the facilities to be used at each performance site listed in
item 9. Face Page, and briefly indicate their capabilities, pertinent
capabilities, relative proximity, and extent of availability to the project. Use
"Other" to describe the facilities at any other performance sites listed in item
9 on the Face Page and at sites for field studies. Use continuation pages if
necessary. Include an explanation of any consortium/contractual arrangements
with other organizations.

[X] Laboratory: Harold Craighead's laboratory has the capabilities to evaluate
and test optical components and systems. Cornell University has extensive
facilities and equipment for optical device fabrication research, prototyping
and testing. The National Nanofabrication Facility (NNF) provides resources and
technical expertise to researchers in microstructure science. Extensive
experience also exists in fine feature patterning of semiconductors and optical
materials. Unique capabilities exist for integration of electron beam
lithography and ion etching for novel thin film fabrication. The NNF is located
in the Knight Laboratory, specifically designed and construed for nanostructures
research, on the Cornell University campus. The cleanroom facilities comprise
7,500 sq. ft. of approximately Class 500 area, including Class 10 local
processing benches within that area. Particular care was given during
construction to vibration and electromagnetic isolation of the advanced
lithography facilities. The Materials Science Center at Cornell provides
computational facilities and extensive electron microscopy and thin film
capabilities.

[_] Clinical:

[_] Animal:

[_] Computer:

[_] Office:

[_] Other( ):

--------------------------------------------------------------------------------
ADDITIONAL INFORMATION Provide any other information describing the environment
for the project. Identify support services such as consultant, secretarial,
machine shop, and electronics shop, and the extent to when they will be
available to the project.

HH Principal Investigator/Program Director (Last, first, middle)
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

RESOURCES AND ENVIRONMENT

(Soane Biosciences Inc.)
--------------------------------------------------------------------------------

FACILITIES. Mark the facilities to be used at each performance site listed in
Item 9, Face Page, and briefly indicate their capabilities, pertinent
capabilities relative proximity, and extent of availability to the project. Use
"Other" to describe the facilities at any other performance sites listed in Item
9 on the Face Page and at sites for field studies. Use continuation pages if
necessary. Include an explanation of any consortium/contractual arrangements
with other organizations.

[X] Laboratory: Soane BioSciences (SBio) was formed as a division of Soane
Technologies (STI) in 1993, and was spun-off as an independent, venture-capital
backed company in 1995 to develop and commercialize advanced electrophoresis
media and devices. SBio currently has 12 employees and is located in a 6,000 sq.
ft. facility in Hayward, California, about 70% of which is lab space. Adjoining
SBio is STI's 6,000 sq. ft. facility, providing additional equipment and
resources freely available to SBio.

SBio's labs are well-suited for organic and polymer synthesis, with a total of 6
chemical fume hoods and extensive synthesis equipment. SBio also has extensive
equipment for performing material characterization, electrophoresis and
molecular biology experiments. SBio's labs and offices are equipped with a
total of 12 PC's (pentium and 486 model), networked with Novell software.

[_] Clinical:

[_] Animal:

[_] Computer:

[_] Office:

[_] Other( ):

Bioanalytical and Molecular Biology Equipment
SBio has a Pharmacia A.L.F. DNA sequencer, a Beckman P/ACE 2100 capillary
electrophoresis system with UV detection (a laser-induced fluorescence detector
is on order); vertical and horizontal electrophoresis units, including a manual
DNA sequencer, with power supplies and visualization equipment; equipment for
preparation of DNA sequencing samples including thermocycler, microcentrifuge,
and centrifugal evaporator; along with other miscellaneous items including a
Millipore Milli-Q Plus ultra-pure water system, freezers, refrigerators,
glassware, etc.

Monomer and Polymer Synthesis and Characterization Equipment
SBio has extensive polymer and organic synthesis equipment including reaction
kettles, homogenizer, temperature controllers, rotary evaporators, vacuum ovens,
centrifuge, filtration equipment, freeze dryer, UV curing lamps and other
miscellaneous items (balances, pH/conductivity meter, etc.) For analysis and
characterization, SBio has a Hewlett-Packard 1090 HPLC with both diode array and
refractive index detection. Brookfield viscometer with temperature control,
Nikon visible microscope with CCD camera, differential scanning calorimeter, and
a refractometer.

Machine Shop
SBio has access to STI's machine shop with state-of-the-art equipment including
CNC mill, which will be used for this project.

HH Principal Investment/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------
RESOURCES AND ENVIRONMENT
(The Whitehead Institute)
--------------------------------------------------------------------------------

FACILITIES: Mark the facilities to be used at each performance site listed in
Item 9. Face Page, and briefly indicate their capacities, pertinent
capabilities, relative proximity, and extent of availability to the project. Use
"Other" to describe the facilities at any other performance site listed in Item
9 on the Face Page and a: sites for field studies. Use continuation pages if
necessary. Include an explpanation of any consortium/contractual arrangements
with other organizations.

[X] Laboratory: There is 16,500 square feet of laboratory space divided between
the 4th and 5th floors of The Whitehead Institute/MIT Genome Center. These
include bench space, shared rooms for radioisotope handling, consistent
temperature (4 degrees C and 37 degrees C), darkrooms, and heavy equipmemt
rooms.

[_] Clinical:

[_] Animal:

[X] Computer: The Center currently has a network of UNIX workstations (7 DEC
workstations, 7 SUN workstations, and 4 new DEC Alphas) and Macintosh personal
computers connected through Ethernet. Outside connections provide full access to
computer services at The Whitehead Institute (e.g. MedLine) as well as to other
sources of information available at MIT and over the world-wide Internet. Also
available is a PC set-up for automatic visual entry of dot blot data with CCD
Camera and a Montage film recorder.

[X] Office: There is approximately 5500 square feet of office space on the 5th
floor of the Genone Center. Available to me are Ricoh photocopy machines, Cannon
FAX machines, IBM typewriters, secretarial support, file cabinets and storage
space.

[_] Other ():

--------------------------------------------------------------------------------
MAJOR EQUIPMENT. List the most important equipment items already available for
this project, noting the location and pertinent capabilities of each.

There are low and medium speed centrifuges, preparative ultracentrifuges,
scintillation counters, incubators for bacteria, liquid nitrogen freezers,
laminar flow hoods, microscopes, electrophoresis and sequencing facilities and
PCR machines available.

--------------------------------------------------------------------------------
ADDITIONAL INFORMATION. Provide any other information describing the environment
for the project. Identify support services such as consultant, secretarial,
machine shop, and electronics shop, and the extent to which they will be
available to the project.

We have available to us the resources of The Whitehead Institute, including an
electron microscopy laboratory, a florescence photomicroscope, an histology
laboratory, polynucleotide and polypeptide synthesis facilities, a P3
laboratory, a library and computer facility. Chemical storage, media preparation
and a glassware washing facility are located on the 4th floor of the Genome
Center. Also available to us is access to the resources of the MIT Biology
Department.

Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------
1. Specific Aims

In this proposal, we describe a three-year project to develop, integrate and
distribute into the Human Genome Program (HGP) pilot sequencing projects a
fluorescent sequencing instrument and software system capable of meeting the
logistic and cost demands of sequencing the entire human genome. This project
consists of taking a system level approach to developing methods,
instrumentation and software designed from the start to work within, and be
proven in, the framework of genome laboratories. These aims will be met by
building upon the high-throughput system (version 1.0) developed under NIH
funding at CuraGen. This program will produce two sequential improvements:

. version 1.1, for pilot scale sequencing [XXXXXXXXXXXXXXXXXXXXXX]

. version 2.0, for full scale genome sequencing [XXXXXXXXXXXXXXXXX
XXXXXX]

Project Overview

MIT Whitehead Genome Center
-----------------------------------
Automation Workflow Informatics
-----------------------------------------------------
Sample Prep Finishing
Autoloading Assembly
---------------------------------------------------
SBio NNF
Media Optics
---------------------------
Electrophoresis BaseCalling
system
Replicated Substrates
-----------------------------------
---------------------------------------------------

The project duration is designed to parallel that of planned HGP pilot studies,
allow for the use of the systems in these studies, and produce a finished system
ready for distribution to sites selected to carry out full scale human genome
sequencing efforts. The project is designed to effect the research tasks
necessary both to achieve the requisite technical objectives as well as to
create the track record necessary to make this the system of choice for wide
scale genome sequencing.

As indicated by the diagram above, CuraGen, Soane BioSciences (SBio), and their
academic collaborators at MIT/Whitehead and the Cornell National Nanofabrication
Facility (NNF) are taking a modular phased approach built upon existing and
proven technology. The evolving CuraGen/SBio DNA analysis system has been
developed and "road tested" in a production sequencing environment at CuraGen.
Since July 1995, it has been in place at the Whitehead Institute. This project
builds on the NIH and DOE funded efforts at each of the four groups. The P.I.
Dr. Went will coordinate, integrate and focus the actives of the individual
groups and rely on his interdisciplinary team at CuraGen for instrument,
software and system development. The experiences of Drs. Trevor Hawkins and
Lincoln Stein at Whitehead in automation, workflow and informatics is brought to
bear to insure dove-tailing of the instrument development into existing
infrastructure. The expertise of Drs. Herb Hooper, David Soane and the Soane
BioScience team in advanced media and replication enables the development of
advanced reusable and disposable separation systems. Leadership in the field of
microfabrication, with an emphasis on microfabricated optics, is provided by Dr.
Harold Craighead at the Cornell NNF.

[Confidential Treatment Requested]

Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------

We begin with the system in routine operation at CuraGen and make the following
specific improvements to produce advanced generations: version 1.1 (by October
1997) and version 2.0 (by April 1999).

Component (Performance Site) Specific Aim

Sample preparation(MIT, CuraGen) Develop and automate solid phase loading and
purification methods to remove the bottleneck at
the interface between existing robotic sample
preparation and loading of fluorescent
electrophoretic systems and to reduce the overall
reagent costs (v. 1.1).

Develop novel bi-directional sequencing protocols
(v. 1.1, 2.0).

Electrophoresis system (CuraGen, SBio) Produce a low cost, robust electrophoresis system
with a disposable, ready-to-run microchannel
(microreplicated) separation sub-assembly (v. 2.0).

Media (SBio) Develop advanced separation media for multiple runs per
gel (v. 1.1).

Develop dynamic porosity media to extend read lengths
(v. 1.1, 2.0).

Develop media for disposable, microreplicated gel
assemblies (v. 2.0).

Detection (Cornell): Implement a binary detection system for use with the
microreplicated separation system (v. 2.0).

Informatics (CuraGen, MIT) Tie electrophoresis instruments into laboratory work-flow
(v. 1.1).
Produce sequencing data with explicit confidence levels
for each base call (v. 1.1).
Enable 8-dye protocols (v. 1.1, 2.0).
Enable automated assembly and statistically qualified
finishing of sequences (v. 2.0).

2. Background and Significance

The human genome comprises three billion base pairs of DNA. In order to sequence
the entire genome, a phased approach has been implemented by the NIH and DOE.
The first requirement is the production of a conceptual array of large
contiguous DNA sequences (large insert clones) spanning the 23 chromosomes.
These "physical maps" must accurately represent the human genome and be in a
form amenable to DNA sequencing. To make these physical maps amenable to
sequencing, the individual large-insert clones will be further sub-divided into
templates (either physically by subcloning or by primer walking) for direct DNA
sequencing. Once the precise sequence of these templates is determined (by
Sanger sequencing and fluorescent detection) they will be "assembled" into
accurate ("finished" sequence) virtual representations of the original
large-insert clones. The sequences of these large-insert clones are then
assembled to form the full finished sequence of the human genome.

2.1 HGP: Technical Challenges

The challenge, constrained by economics and time, is to produce a finished
genome sequence at a cost of under $0.10 per base at a rate of 600 Mb per year,
in order to complete the genome for under $300 million dollars in a five-year
period. Physical maps of the genome are now estimated to contribute less than 1
penny per base to the finished sequence cost (Lander et al., 1995) and to be
nearly complete in time for the start of large scale sequencing of the human
genome. This leaves $0.09 per base to go from the physical map to assembled and
finished sequence. The most successful high-throughput DNA sequencing centers in
the world are currently

Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------
producing C. elegans sequence from physical maps at $0.50 per finished base at a
rate of 10 Mb/year (Sulston et al., 1992; Wilson et al., 1994 ). The rate and
cost of sequencing complete bacterial genomes and yeast chromosomes is
comparable. To meet the HGP goals, it is necessary to increase the rate of
production of finished sequence 60-fold while decreasing the cost 5-fold over
these current projects.

The logistical concerns of genome-scale DNA sequencing are essentially those
derived from the need to increase throughput and reduce cost. This can only be
done by improved technology integration and automation. Historically, the
introduction of fluorescent four color sequencing into the life science research
market enabled the sequencing of individual clones, small contiguous regions,
and, when pushed to the limits of the original technology, the sequencing of the
first complete bacterial genome (Fleischmann et al., 1995). An early 4-dye
commercial instrument produced by Applied Biosystems, Inc., the ABI 373, and its
subsequent replacement, the 377, were not designed with the logistics of large
scale genomic sequencing in mind. Specifically, these instruments were not
designed to efficiently integrate into a "factory environment" consisting
primarily of robotic sample handling automated within an informatics framework.

Producing finished sequence at 600 Mb per year or 2 Mb per day requires the
production of upwards of 40,000 template and sequencing ladders a day (based on
500 bp reads/sample and 10 fold redundancy). Pilot programs are currently aimed
at achieving 1/10th of this rate. The workflow, upstream robotics and
informatics that are being planned and currently developed for pilot studies
(Lander et al., 1995), if properly assisted by a new generation of sequencing
technology such as the v. 1.1 system proposed here, would be capable of
processing 4,000 samples per day. With the advent of the v. 2.0 system, these
pilot programs could be scaled to meet the expected 40,000 sample per day
throughput required for a large scale sequencing effort. In order not in any way
--------------
to duplicate the upstream workflow and robotics efforts, we concentrate in this
-------------------------------------------------------
proposal on developing advanced sequencing instrumentation and systems to
dove-tail into the upstream capabilities. This represents an approach in which
increases in lane density, electrophoresis speed, and sensitivity of a
fluorescent sequencing instrument are integrated into existing front-end
robotics to yield the requisite targeted "system level" throughput increases and
concomitant cost reductions.

2.2 HGP: Cost Analysis

Cost analysis studies are regularly used to determine which problems to work on
from a return on investment standpoint, and which components of a problem are
sensitive to cost reductions. Recent examinations of the Human Genome Project
(HGP) have led to a separation of the project into two phases. During the first
phase, scheduled to last three years, additional technology will be developed
and pilot studies will be conducted to provide a solid basis for determining how
best to carry out the full scale project during the subsequent five year second
phase.

Technology improvements are often driven by the need for better cost
performance. In the computer industry this has lead to a doubling of potential
with a concomitant halving of cost every 18 months. This has become known as
Moore's law, in recognition of its major proponent, Gordon Moore of Intel
Corporation. With this in mind we have investigated the impact that advances in
the major technology components would have (primarily) on the sequencing portion
of the HGP. Ultimately, the impact of the HGP will be judged by the number of
genetic disorders that can be more effectively diagnosed and treated, which will
require many-fold genomic sequencing.

Physical mapping and sub-cloning efforts, which are planned prerequisites of
large scale sequencing are expected to contribute from $.01 to $.03 to the cost
per base of finished sequence. (Throughout this discussion we have adapted the
NCHGR convention of using the finished base as the unit of measure to allow a
comparison of the impact of technological advances on cost.) Pilot projects are
better suited to address these costs and we therefore concentrate our efforts
here on improved sub-clone sequencing and assembly to produce a finished
sequence.

A sequencing cost analysis (Table 2.1; see Appendix 2 for a breakdown of the
analysis and supporting assumptions) highlights the major contributions to the
final cost, which can be reduced by improvements in the following technology
areas: sample preparation and loading, electrophoresis systems, separation
media, detection and informatics. Specifically, the two largest contributions to
the finished cost per base are (1) materials for template preparation and
sequencing reactions and (2) labor costs associated with sequence assembly. The
aggregate sensitivity and bi-directional sequencing improvements described
herein have the potential to reduce this cost 3-fold, while the addition of
confidence information in base calling, and accurate lane tracking provided by
micro-channels would enable the automation of the assembly and lane-finding
processes, and another 3-fold decrease in cost. This analysis further indicates
that the expected improvements in automation being developed in pilot studies
are sufficient to meet the overall goals, and do not need to be undertaken here.

Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------
In addition to financial issues, these calculations provide additional
information on other resource and labor issues that may contribute to logistical
problems. These specifically include the demand existing technology presents for
pouring and handling over 1000 gels a day and the space and power requirements
to house over 250 sequencing instruments. From this information, it is clear
that disposable, ready-to-run gels will tremendously decrease the logistical and
financial burdens of gel-pouring.

These improvements taken together can yield a cost saving based on using
commercially available systems of over $800 million. Using the expected
investment of approximately $80 million for the first phase of the human genome
project (over the next 3 years), this 10-fold return on investment would yield
in the ensuing 5 years a 50% compounded return on investment. This analysis
clearly indicates the importance of the pilot programs and an emphasis on
technology development for the success of the humane genome project. This
project aims to address those critical components highlighted above, and to
deliver a system to cost-effectively sequence the genome.

Table 2.1 Cost analysis for sequencing the human genome.

Advanced Commercial [XXXXXXXXX] [XXXXXXXXX] [XXXXXXXXX]
------------------- ----------- ----------- -----------

Project cost summary:
--------------------
Total Fixed costs $54,594,000 [XXXXXXXXX] [XXXXXXXXX] [XXXXXXXXX]
Total Material Costs $273,600,000 [XXXXXXXXX] [XXXXXXXXX] [XXXXXXXXX]
Total Labor Costs $329,550,000 [XXXXXXXXX] [XXXXXXXXX] [XXXXXXXXX]
----------------- ------------ ------------ ----------- -----------
Total project cost $657,744,000 [XXXXXXXXX] [XXXXXXXXX] [XXXXXXXXX]

Cost per finished base:
Fixed cost/finished base $0.018 [XXXXXXXXX] [XXXXXXXXX] [XXXXXXXXX]
Material cost/finished base $0.0912 [XXXXXXXXX] [XXXXXXXXX] [XXXXXXXXX]
Gel related labor cost/finished base $0.0139 [XXXXXXXXX] [XXXXXXXXX] [XXXXXXXXX]
Sample labor cost/finished base $0.0160 [XXXXXXXXX] [XXXXXXXXX] [XXXXXXXXX]
Sequence assemble labor cost/finished base $0.0800 [XXXXXXXXX] [XXXXXXXXX] [XXXXXXXXX]
------------------------------------------ ------- ------- ------- -------
Total sequencing cost/finished base $0.2192 [XXXXXXXXX] [XXXXXXXXX] [XXXXXXXXX]

Totals with 70% Indirect cost on labor and materials:
----------------------------------------------------
Total project cost $1,025,355,000 [XXXXXXXXX] [XXXXXXXXX] [XXXXXXXXX]
Total sequencing cost/finished base $0.3418 [XXXXXXXXX] [XXXXXXXXX] [XXXXXXXXX]
Physical mapping and sub-cloning $0.0333 [XXXXXXXXX] [XXXXXXXXX] [XXXXXXXXX]
-------------------------------- ------- ------- ------- -------
Total cost/finished base $0.3751 [XXXXXXXXX] [XXXXXXXXX] [XXXXXXXXX]

Summary of Resource needs:
-------------------------
Gels to set up per day 1,108 [XXXXXXXXX] [XXXXXXXXX] [XXXXXXXXX]
Total Seq Units needed to meet quota 277 [XXXXXXXXX] [XXXXXXXXX] [XXXXXXXXX]
Number of people necessary 1,373 [XXXXXXXXX] [XXXXXXXXX] [XXXXXXXXX]
Estimated Sq. ft 186,265 [XXXXXXXXX] [XXXXXXXXX] [XXXXXXXXX]

2.3 Component Technologies

The process of sequencing the genome leads to the production of small (less
than 2 Kb) fragments of DNA from large-insert clones (BACs, YACs, P1s). Sanger
sequencing ladder products can be generated from these fragments by a number of
robotic means, with final product residing in the wells of a microtiter dish (96
or 384 well). This process is executed now at major genome facilities. At this
point, a second stage of systems for performing electrophoresis, fluorescent
sequencing, and sequence assembly take over. This project replaces the second
stage, enables new Sanger protocols, and carries through to use the host site
informatic system to deposit automatically the finished sequence into a
database.

2.3.1 Sample Preparation/Loading

The output of most Sanger protocols is a mixture of input DNA, fluorescent DNA
ladders, and unreacted fluorescent primers or nucleotides. In order to prepare
the sample for electrophoresis, it must be purified. The

[Confidential Treatment Requested]

Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------

use of solid phase techniques for this purification step has increased
dramatically over the last few years as more biochemical methods are adapted for
use with magnetic particles (Uhlen, 1989; Hultman et al., 1989; Hultman et al.,
1991; Hawkins, 1994). The advantages of using magnetic particles are based upon
the ease with which DNA can be manipulated once immobilized on the magnetic
particles. In theory, the need for centrifugation stages could be removed from
molecular biology if DNA fragments were fixed either temporarily or permanently
to the magnetic solid phase. However, the use of a solid-phase has not yet been
successfully applied to the automated loading, a process that could benefit from
the simplicity and throughput increases associated with the coupling of the
transport and purification advantages of solid-phase systems.

2.3.2 Gel Electrophoresis

There are several important features of a gel electrophoresis system: (1) the
interfaces for gel and sample loading, (2) throughput, i.e., the number of
samples (and bases) that can be processed per unit time, (3) read length, (4)
sample volume required, and (5) the back-end interface for fragment detection.
Traditionally, electrophoresis has been carried out between two plates of glass
(or plastic) separated by a spacer, with a polymeric medium to sieve the DNA.
Commercial state-of-the art is now the ABI 377 at 36 sample/450 bases/2.5 hrs.,
providing 6.5 Kb/hour of raw sequence. Performance has been improved using thin
slab gels, capillary arrays, and microchannel devices.

Thin slab gels. One of the primary factors in determining electrophoresis time
--------------
is the ability to apply high voltages, generating significant heat, without
significant temperature gradients. Smith and coworkers (Brumley and Smith, 1991)
pioneered (less than) 100 (um)m gel widths and established that 10 bp/min can
be attained at 200 V/cm. Vertical and horizontal thin gels have been used, but
the horizontal approach has significant advantages from the standpoint of
automating sample loading. CuraGen has adapted the horizontal thin gel approach
with traditional gel and sample loading to produce a routine and automatable
device (see Section 3).

Capillary array electrophoresis. Introduced to DNA sequencing by Mathies and
-------------------------------
coworkers (Huang et al., 1992a,b), CAE combines the high-speed separation
attainable in individual capillaries with the throughput provided by
multiplexing. Several groups are actively developing capillary array systems
(Takahashi et al., 1994; Ueno and Yeung, 1994; Barker, 1995), including at least
three commercial entities. In addition to throughput, CAE offers the potential
for automated gel and sample loading, which is now being realized with the
recent advances in replaceable matrices. Lower viscosity matrices are still
needed, as are more stable wall coatings. Electrokinetic injection is
inefficient with respect to sample utilization, requiring a concentrated DNA
sample of which only a fraction is injected. The detection region of capillary
arrays is ultimately determined by the capillary outer diameters, an inherent
limitation for high lane densities.

Microchannel gel devices. Miniaturized separation lanes and devices can be
------------------------
fabricated in planar substrates using micromachining techniques. Terry et al.
(1979) microfabricated a gas chromatograph in silicon sixteen years ago,
providing the basis for a commercial, portable GC manufactured by Microsensor
Technologies Inc. Microfabricated electrophoresis devices were proposed by
several groups around 1990 (Kovacs et al., 1989; Pace, 1990; Manz et al., 1991;
Harrison et al., 1992a). Several reports have demonstrated sample injection
schemes, high speed electrophoretic separations of dyes and labeled amino acids,
and pre- and post-column reactions (Harrison et al., 1992b; Jacobson et al.,
1994a-c). Recently, rapid, high-resolution separations of dsDNA (Wooley and
Mathies, 1994), oligonucleotides (Effenhauser et al., 1994) and DNA sequencing
ladders (Balch et al., 1994; Went, 1995) have been reported. The revolutionary
vision is to create fully-integrated chip-sized devices with on-board sample
preparation, followed by automated injection, separation and detection. While
this goal appears fundamentally achievable, it presents significant technical
risk in the 3-5 year time frame. The more evolutionary approach is to
microfabricate the separation device, and utilize continually improving robotic
methods for sample preparation and loading. We have taken the second approach,
utilizing the modular design of CuraGen's horizontal thin gel system (Section
3). This device can be integrated with microfabricated front-ends as they become
available.

2.3.3 Electrophoretic Separation Media

The separation matrix is a critical element of the separation platform in DNA
sequencing. The use of crosslinked polyacrylamide in slab gel sequencing has
changed little since its introduction in the late 1950's . The disadvantages of
in-situ acrylamide polymerization are well-known, including monomer toxicity,
labor intensity, and reproducibility issues. These effects become logistically
untenable at the 40,000 lane (1000+ gel) per day level. Pre-cast gels, popular
in a number of electrophoresis applications, have not been introduced for DNA
sequencing. In contrast to slab gels, there has been significant innovation in
media for capillary electrophoresis, centered around the use of uncrosslinked
polymer solutions as replaceable matrices. Since the early reports by Zhu et al.
(1989) and Chin and Colburn (1989), a number of groups have demonstrated that a
wide variety of uncrosslinked polymer solutions can be used for separating dsDNA
fragments (e.g., Heiger et al., 1990;

Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------
Grossman and Soane, 1991; Pulyaeva et al., 1992). These dsDNA matrices typically
have low viscosities and can be routinely filled and replaced at low pressures.

The development of replaceable matrices for DNA sequencing has been more
difficult, as higher concentration, higher viscosity solutions are required. In
1993, Karger and coworkers (Ruiz -Martinez et al., 1993) reported the use of a
6% uncrosslinked polyacrylamide (PAA), which provided single base resolution to
350-400 bases. Since then, additional reports on the use of uncrosslinked PAA
(Grossman, 1994) and other polymers (Johnson et al., 1995; Yeung et al., 1995)
as replaceable matrices for DNA sequencing have appeared. These matrices are
still relatively viscous, typically requiring several hundred psi and several
minutes for loading into standard (e.g., 75-100 (mu)m ID) capillaries. Matrix
loading in microfabricated lanes is likely to be more difficult, and existing
reports on sequencing in microfabricated devices have employed in-situ
polymerized gels (Balch et al., 1994; Went, 1995), similar to the early work in
capillaries.

2.3.4 Fluorescent Detection

Fluorescent detection of 4-dye Sanger fragments from a single lane was pioneered
by Smith and coworkers (Smith, 1986) and is currently available only from
Applied Biosystems. In its early and current embodiment, a scanning
laser/detection unit rasters across a slab gel simultaneously exciting a
detecting fluorescence from four select dye labels. Filters, initially employed
on a rotating wheel, have been replaced with a more efficient reflective
grating, allowing for the detection of 1.5 (upsilon)l of a standard Sanger
ladder.

Scanning. As capillary arrays are employed, scanning systems have been improved
--------
to contain confocal illumination and detection (Mathies and Huang, 1992) to
reduce scattering off the glass. These systems are very efficient and have been
commercialized for non-sequencing use. Their use for imaging slab systems is
also growing (Brumely, 1994). Sample volumes that are actually required for
robust detection are less than 100 nl (Smith, 1993), 15-fold more sensitive than
the ABI system.

2.3.5 Informatics: Image Processing, Base Calling and Sequence Assembly

Determination of DNA sequence from fluorescent images involves the following
steps: lane finding and identification, background elimination, spectral matrix
conversion, base calling, and analysis/editing . At the 36 sample per day level,
this process involves a significant amount of human intervention for lane
calling and sequence editing. At 40,000/day, the system must permit automated
transfer throughout with under 1% manual intervention. Existing automated lane
finding software is capable of calling only 90% of sequencing gels without human
intervention (Stein, pers. comm.). Tibbetts and coworkers (Golden, et al., 1993;
Tibbetts, 1993) have applied neural networks to the base calling problem, with
significant improvements over traditional methods. Giddings (1993) has also
worked on this problem using expert modules which judge peaks to evaluate the
confidence of base calls.

Unlike all other stages of the process, assembly and finishing have
traditionally required considerable human attention including hours of attention
at a computer screen and custom-designed experiments to resolve uncertainties.
More than 60% of the costs of large scale sequencing projects may go to assembly
and finishing, due to their low efficiencies. There have been considerable
advances in sequence assembly. Specifically, PHRAP (Phil Green, pers. comm.) and
FAK (Gene Myers, pers. comm.) represent substantial improvements over previous
tools and essentially solve the issues of assembly with few repeats, yet still
have serious difficulties with many mammalian sequences.

[Confidential Treatment Requested]

Figure 3.1 DNA analysis system overview [XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]

In this section, we review development at each of our four groups which provides
the foundation for this project. These preliminary results are organized by
component technology rather than group, consistent with our systems approach.
The starting point of this project is CuraGen's current version 1.0 DNA analysis
system, shown in Figure 3.1. As an end user requiring high throughput,
integrated DNA analysis systems, CuraGen has developed

[Confidential Treatment Requested]

Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

a horizontal thin gel electrophoresis system with a proprietary imaging
spectrograph adapted to DNA sequencing, ready for integration into automated
systems. The optical schematic shown in Figure 3.1 highlights the principle
hardware components of CuraGen's DNA analysis system. This device has been in
operation at CuraGen since early 1993 for a variety of applications, including
sequencing (see Figure 3.1 and A.0 in the Appendix for representative M13
results). The system has several advantages:

. It is a high speed modular device that is adaptable and
upgradeable. Current speeds are 700 bph (bases per hour) with read
lengths of 450bp typical, as shown in Figure A.0. In general, we
load 1/15th of a standard ABI sample. The system is running at 36
lanes per gel at 14 Kb of raw sequence/hr.

. Full spectrum detection under software control allows any dyes to
be used (see Figure 3.3). We have demonstrated the feasibility of
five colors being used simultaneously (four for sequencing, the
fifth for reverse sequence or fingerprint information).

. It includes trainable image analysis and base calling software. In
collaboration with Dan Seligson of Intel, CuraGen has developed a
modular set of algorithms for preprocessing and base calling. The
advantages of the software are full UNIX compatibility, a database
access tool set (with easy i/o) and high processing speed (less
than 5 secs/lane on an Intel P90).

The ability to integrate this device into automated systems, both in terms of
front end sample handling and back end image processing and base calling, makes
this system a cost effective alternative to the ABI.

3.1 Sample Preparation (MIT, CuraGen)

3.1.1 Automated Protocols for Large Scale Genome Sequencing (MIT)

Work at MIT/Whitehead has culminated in a second-generation automation system
for purification of templates and production of sequencing ladders. The current
"Sequatron" system has a throughput of 80 microtiter plates per 24 hours,
starting with grown M13 supernatants for DNA isolation or plates of PCR products
and ending with completed pooled sequencing reactions ready for denaturing and
loading on a gel.

3.1.2 Solid phase technology /Automated Loading/Concentrating (MIT, CuraGen)

We have developed a simplified system for the manipulation and isolation of DNA
using solid phase purification that does not rely on streptavidin-biotin,
digoxigenin-antidigoxigenin or sequence-specific interactions. We refer to this
as Solid-Phase Reversible Immobilization (SPRI, Hawkins et al., 1994). The
method provides a solid-phase purification procedure that can be used for DNA
extraction, purification and concentration. These methods have been introduced
into routine use in the Whitehead DNA Sequencing Core.

[GRAPHIC APPEARS HERE]

Figure 3.2 Trace file obtained from direct SPRI loading experiment performed
at MIT on a CuraGen device. Sequence of M13 obtained using standard
ABI kit. Trace shows bases 100 - 200 taken over a 7 minute stretch.
Applied voltage was 5 KV over 25 cm, 200 V/cm.

The isolation of extension products using solid phase support has had
limited use, mostly due its lack of applicability to cycle sequencing, the
elevated cost of magnetic particles and the lack of demand for replacements to
ethanol precipitations. A key advantage of the SPRI procedure is that the
isolation step functions as a concentrating step and can be directly
incorporated into an automated loading step. We found that SPRI can both
concentrate dye-primer reactions as well as clean-up dye terminator reactions
prior to electrophoresis. This removes the need for ethanol precipitation or
spin columns. Of great importance, the primers do not have to be derivatized
---
with haptens such as biotin or digoxigenin to be subsequently isolated using
SPRI, and hence it is applicable to amplification based sequencing techniques,
now the methods of choice in the genome community. Direct loading with SPRI
beads into formamide wells (see Figure 3.2) allows the precise control of the
loading and motivates the design of an automated system.

One principal advantage of the CuraGen DNA Sequencer is the ability to image
many distinct dyes simultaneously. Methods for producing sequencing ladders from
both directions have been tested, and to date we have shown that 5- dye
sequencing can be achieved with the fifth color being used to obtain a T track
from the opposite end of the sequencing template (Figure 3.3). Strategies
employing more then 4 dyes have utility in fingerprinting, assembly and
finishing. Combined with Figure 3.4, this clearly establishes (1) the
feasibility of 5-dye protocols for alignment (using far distant red-dyes) and
(2) the potential to sequence in both directions using 8 dyes. With this proof
of principle and our current advances in fluorescent detection, we are in a
position to develop full bi-directional sequencing strategies.

[GRAPHIC APPEARS HERE]

Figure 3.3 Bi-directional sequencing. The trace shown in 3.3a is a
sequence off the forward primer of a sequamark template, while
trace 3.3c is the same template sequenced from the reverse primer.
The center trace 3.3b is the result of a sequencing run from a
single tube in which two different primers, FAM-forward and
JOE-reverse, were placed with the ddT mix. The resulting product
terminates in both directions with two different dye labels. A
color copy of this figure is included in the Appendix.

[LINE GRAPH APPEARS HERE]

Figure 3.4 Spectral emission measured from spatial cross section taken
from electrophoresing dye-labeled primers. All 8 have been tested
and the individual dyes are easily resolved. A 5-dye set can be
chosen and we are currently evaluating 8 dye possibilities. A color
copy of this figure is included in the Appendix.

[Confidential Treatment Requested]

Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------
[XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]

3.2 Gel Electrophoresis (CuraGen)

We have developed a flexible thermal regulation box (air, water, and passive)
for the use and testing of a variety of thin slab and microfabricated channel
devices (see Figure 3.1). It requires under 10 minutes of assembly time and
yields bubble-free, high quality 50 (mu)m gels over 95% of the time.
Representative capabilities are shown in Figure A.0, 3.2 and 3.3, in which raw
data (i.e., matrix converted without any filtering) clearly indicates the
ability to load, electrophorese and detect from these gels.

3.1.1 Routine Thin Gel Capabilities.

CuraGen has logged over 7000 lanes on its thin slab system and currently has 4
systems in place. During the month of August 1995, MIT carried out over 500
lanes with comparable results.

Multiplicity (36). Figure A.0, 3.2 and 3.4 taken from 36 lane runs using
sharkstooth combs. Manual lane identification easily picks separate lanes.

Length of Read (500). Figure A.0 shows a sequence of M13 at 450+ bp.

Speed (1 hr). The results shown in Figure 3.2 from a SPRI experiment
run at MIT clearly show that at 5kV, we can obtain base pair resolution to
400 bases at 17 cm from the loading region at a rate of 15 bp/min.

3.2.2 Microchannel Substrates

Figure 3.5 shows a trace taken from a 96-channel glass substrate used to test
microchannel electrophoresis in which 10 nl of input Sanger ladder was loaded.
Although the quality is less than a comparable run on a thin slab, the sequence
was correctly called. Improvements to the loading (larger volumes) and imaging
capabilities are being developed under a NIH Phase II SBIR.

[GRAPHIC APPEARS HERE]

Figure 3.5 Processed sequencing data obtained from sequencing an M13 template
in two different configuration: (a) top, short thin slab with laser
10 cm from loading region; (b) bottom, glass microchannels with
laser 10 cm from loading region. Color copies of this figure are
included in the Appendix.

3.2.3 Polymeric Substrates

Mass production of polymeric substrates via molding or casting will be
significantly less expensive than etching glass, will not produce acid waste,
and will therefore enable our strategy of providing ready-to-use, disposable
microchannel substrates. Moreover, there are a great number of methods for
fabricating plastics and a variety of transparent materials to employ. The
personnel at SBio have investigated the polymerization and optical
characteristics of a variety of plastic materials. We have a long track record
of replicating the surface and bulk features of molds in plastic with high
precision, as will be required for microreplication of lanes in plastic. In
addition, our experience with mass producing such substrates in a commercial
setting will enable the eventual production of the proposed microreplicated
separation subsystems.

3.3 Advanced Separation Media (SBio)

SBio is developing improvements in replaceable gels for DNA sequencing by
capillary electrophoresis (CE), as reviewed below. These preliminary results
demonstrate SBio's expertise in gel media, and a technology base that serve as a
foundation for this project.

[Confidential Treatment Requested]

Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------
1st generation replaceable matrices. SBio is developing non-crosslinked,
replaceable matrices for CE based on N-substituted acrylamide polymers, which
are known to provide an order-of-magnitude increase in hydrolytic stability
relative to polyacrylamide (Righetti et al., 1993). Hydrolysis of the amide
bonds in polyacrylamide (PAA), which is accelerated at high pH and urea levels,
will limit the shelf life of replaceable matrices based on uncrosslinked PAA. We
have developed copolymer formulations of N-substituted acrylamides that provide
resolution comparable to PAA matrices (Figure 3.6), while simultaneously
providing enhanced stability and shelf life. We have also developed
reproducible, scaleable synthesis and purification processes for these matrices.
We are currently optimizing matrix formulations to reduce viscosity and
electrophoretic run time. The matrix used in Figure 3.6 was loaded into the 65
cm long, 75 (mu)m ID capillary in less than 3 minutes under an applied pressure
of 400psi. We believe that incremental reductions in loading pressure and time
can be realized through optimization of MW and MW distributions.

[GRAPHIC OMITTED]

Figure 3.6 Capillary electrophoresis separation of T-terminated extension
products from M13mp18 template on SBio replaceable matrix.
Experimental conditions: capillary length: 65.0 cm (40.0
effective), DNA injection: 12.0 kV - 15 seconds, run voltage: 12.0
kV. M13 sequence prepared via cycle sequencing using labeled primer
and imaged by laser-induced fluorescent detection.

2nd generation replaceable matrices. SBio is developing a novel proprietary
class of replaceable matrices which undergo phase transitions in response to
temperature (Soane and Bae, 1994; Soane, 1994 and 1995), offering the potential
for dramatic reductions in loading viscosity. In these "reversible gels", the
matrix polymer is highly soluble on one side of the transition, and much less
soluble (or insoluble) on the other side. We have demonstrated that this phase
transition property can be utilized to drive a viscosity transition (Figure
3.7), and have used this transition to demonstrate matrix loading in capillaries
under very low pressures, including simple vacuum injection. We have also
developed polymer compositions which provide excellent electrophoretic
properties in addition to this viscosity transition behavior. One family of
compositions is based on a subset of the N-substituted acrylamides which are
temperature-sensitive. Other, non-acrylamide compositions are also being
developed, along with matrices which have a low viscosity at room temperature
and gel upon heating. These 2nd generation matrices hold the promise for
dramatically improving the process of matrix loading and unloading, which will
be especially important if replaceable matrices are to be used in very small
channel microfabricated devices.

Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

[GRAPHIC OMITTED]

Figure 3.7 Temperature induced phase transition in crosslinked microgel system
(A) and uncrosslinked polymer solution (B), resulting in a
reversible viscosity transition in both cases. The exemplary
viscosity curve (C) is for a microgel system with a transition
around 30C. By modifying the polymer composition, matrices can be
designed with viscosity transitions at temperatures ranging from
room temperature to 95 C.

3.4 Fluorescent Detection (CuraGen, Cornell)

[XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]

3.4.2 Microfabricated Optics (Cornell NNF, CuraGen)

[Confidential Treatment Requested]

Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

Figure 3.8 AFM area scan of a continuously blazed grating using focused
ion-beam milling (Shank et al., 1994)

[XXXXXXXXXXXX]

Figure 3.9 Diagram of a vertical optical interconnect showing the location of
the individual components (Bare et al., 1994)

[XXXXXXXXXXXX]

3.5 Informatics (CuraGen,MIT)

Critical issues in high-throughput genomic sequencing include sample
tracking, work-flow management, image analysis, base calling (speed and
confidence), assembly and finishing.

3.5.1 Image Analysis and Base Calling (CuraGen)

[XXXXXXXXXXXXXXXXXXXXXXX]

Figure 3.10 Base calling algorithm and output. [XXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]

[Confidential Treatment Requested]

Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------
CuraGen has developed a modular set of algorithms for preprocessing and base
calling. The software has several advantages:

. it is trainable to new chemistries, dyes or dye coding strategies,

. it reports quantifiable confidence values on each base called, and

. it is based upon a UNIX development platform and easily integrated in
LabBase and other informatics systems.

There are three main modules: a signal preprocessing module, a pattern
recognition module (i.e., base calling for DNA sequencing), and a proofreading
or postprocessing module. [XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]

3.5.2 Assembly and Finishing (MIT)

Major assembly and finishing packages, including PHRAP and FAK, are in use in
our laboratories. We have written our own software, including MAPPER, which
assembles map contigs based on existing assembly tools with forward and reverse
constraints, and FINISHER, which uses the information from the map contigs to
select further experiments. Using these programs we have evaluated sequencing
strategies on simulated and real data. The ability to quantify the confidence of
individual base calls will set the precedent for allowing this entire process to
be conducted in an automated way, and to maintain the integrity of the data.

3.5.3 Work flow

Over the past two years, the MIT Genome Center's Informatics Core has gained
extensive experience in database and sample-tracking issues, and developed three
major systems: LabBase, Workflow Manager, and BuilderIO. Our laboratory
informatics system has been developed to allow FINISHER to download instructions
to robotic workstations. With the direct incorporation of the sequencing
instrumentation into workflow we will be able to place the instrument into an
automated environment and take advantage of the advanced base calling software.

4. Research Design and Methods

The research program outlined herein builds upon the combined experience of the
collaborators to develop technologies to accelerate the completion of the human
genome sequence as well as to enable more routine genome sequencing. As outlined
before, the HGP has evolved to the point where its completion is tractable. This
project plans to hasten that timeline in stages:

1) Version 1.1, a thin slab gel platform addressing the cost and throughput
needs of a 4000 sample/day pilot facility;

2) Version 2.0, a microchannel platform solving the gel and informatics
logistics of a 40,000 sample/day operation, capable of sequencing multiple
genomes and extending high throughput sequencing to other markets in the future.

The project is divided into the five components discussed shown below. Although
primary responsibility for the execution of each sub-task rests with the
collaborating investigators, all advances will be tested in CuraGen's evolving
DNA analysis system. Moreover, each of the project teams will have
instrumentation in place to accelerate the design and testing of individual
components.

A component breakdown with primary responsibilities and delivery dates is shown
below in Table 4.2.

4.0 A Modular Phased Approach

Component Site Deliverable Date

4.1 Sample Preparation MIT/Whitehead v. 1.1 Manual device 4/1/97

[Confidential Treatment Requested]

Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

(Trevor Hawkins) v. 2.0 Autoloading device 10/1/97

CuraGen v. 1.1 5-dye protocol 10/1/96
(Gregory Went) v. 1.1, 2.0 8-dye protocol 4/1/97
4.2 Electrophoresis System CuraGen/Cornell v. 1.1 Electrophoresis station 4/1/97
(Harold Craighead) v. 2.0 Microreplication 10/1/97
prototype

Soane BioSciences v. 2.0 Materials 10/1/97
(Herbert Hooper and v. 2.0 Surface design 10/1/97
David Soane) v. 2.0 Processing 4/1/98

4.3 Separation Media Soane BioSciences v 1.1 Reusable gels 4/1/97
v 1.1, 2.0 Dynamic porosity 10/1/97
v. 2.0 Disposable media 10/1/98

4.4 Binary optics Cornell v. 2.0 Collection optics 4/1/99
4.5 Informatics CuraGen v. 1.1 5-dye base calling 10/1/96
v. 1.1, 2.0 8-dye base calling 10/1/97
MIT/Whitehead v. 1.1, 2.0 Assembly/ 4/1/97
(Lincoln Stein) finishing with confidence
v. 1.1 5-dye assembly 4/1/97
4.6 Systems Integration CuraGen/MIT [XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]
[XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]
[XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]

4.1 Sample Preparation and Loading (MIT, CuraGen)

4.1.1 Manual and Robotic Solid-Phase Sample Concentration and Gel Loading

In preparation for pilot projects, fully automated systems that process DNA
subclones into dye-labeled sequencing ladders have been developed with
throughput capacities of greater than 4,000 samples per day. Our system starts
with the sequencing products (Sanger ladders) presented in 96 or 384 well
bar-coded plates. This approach has three major benefits: (1) it reduces
redundancy with other efforts; (2) it allows new systems to dovetail into
planned pilot studies; and, (3) it reduces the upstream burden by decreasing the
number and the concentration requirements of the sequencing reactions. In
preparation for pilot sequencing, MIT has developed the Sequatron. This device
is currently being tested and used at the MIT Genome Center. We will enhance the
system with the ability to concentrate samples and load CuraGen's DNA sequencing
gels, allowing the integrated system to generate base called sequences directly
from DNA subclones. This enables increased throughput through the continuous
operation of DNA sequencers at lower variable costs.

We will utilize our combined expertise in solid-phase methods, including SPRI
(Hawkins et al., 1994a), biotin-streptavidin (Hawkins, 1994) with photocleavable
biotin derivatives (Olejnik et al., 1995), and digoxigenin-antidigoxigenin
(Hawkins et al., 1994b), to concentrate and purify the final sequencing ladders
produced by sequencing reaction as part of automated gel loading. During the
first year, we will focus on SPRI technology, a highly effective
biochemical/biophysical technique. SPRI uses inexpensive carboxyl-coated
magnetic particles that compose the base material for most magnetic particle
manufacture. The sequencing products are absorbed onto the surface of the
magnetic beads, which are then transferred to the gel using magnetic pins. As an
alternative to magnetic particles, we will also develop a manifold of 12
9mm-spaced pins coated to absorb reaction products directly, allowing washing
and finally elution of products into wells for electrophoresis. This will enable
us to transfer samples from standard 96, 192, or 384-well microtiter plates (9mm
well spacing) into a sequencing gel. Integration of this manifold onto the arm
of a robotic system will allow the full automation of this process.

In conjunction with using coated pins, we will also attempt to use graphite tips
(as used by Tecan and Packard to allow capacitance sensing) to absorb reaction
products and load the gels for electroelution. In addition to automation and
reproducibility improvements, autoloading from a solid substrate enables us to
take advantage of the 15-fold sensitive advantage of the CuraGen device. This
advantage reduces the enzyme utilization requirements at least 10-fold and
translates to a large savings in sample preparation costs.

[Confidential Treatment Requested]

1. Year 1. Modify and improve our solid-phase approaches to enable us to coat 12
channel pins to perform SPRI, biotin-streptavidin capture or
digoxigenin-antidigoxigenin capture and fragment selection.

2. Year 1. Develop a 12 pin loading device to carry out parallel loading from 96
and 384 well plates directly into the wells of the electrophoresis system.

3. Year 1,2. Develop 12 channel pin tools to be fixed to the head of a CRS
articulated robot, as used in our Sequatron system. Set up the robotic system to
enable reproducible loading into wells less than 0.1mm apart. The use of a CRS
articulated arm will give us the necessary flexibility.

4. Year 1,2. Integrate the automated sample concentration and gel loading into
the Sequatron system to provide a fully integrated and automated system.

5. Year 3. Resolve problems of final integration into Sequatron system. Modify
Sequatron software to include collection software of CuraGen electrophoresis
instrumentation.

4.1.2 8-Dye Bi-directional Sequencing

Advances in detection and software allow two reactions, one in each direction,
to be produced from a single template (see Figure 3.2). Initially the reverse
read will be for only one base (5-dye sequencing) for fingerprinting, sizing, or
to enhance assembly strategies. Later evolution to a full 8-dye system will
allow full bi-directional sequencing. [XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXX]

1. Year 1. Establish alternative dye sets for 5-dye and 8-dye sequencing. We
will evaluate the properties (shifting, signal/noise) of a number of publicly
available dyes to expand our current set of 8. Dye-transfer (tested and soon to
be available) dyes (Yu et al., 1995) will also be investigated.

2. Year 1. Build on CuraGen's 2-dye, bi-directional success to develop a kit for
full 5-dye sequencing. The 5th dye will be chosen such that a full 5 basis
matrix transform is robust. This is rather straightforward in a two-laser system
(i.e., no spectral overlap between the standard 4 and the 5th dye).

3. Year 1,2. Reduce enzyme costs by using highly efficient loading protocols.
Sensitivity improvements will minimize the concentration of enzyme needed.
Reaction and cycling conditions will be modified to optimize the results.
Properties of other dyes, including incorporation and detectability, will be
investigated.

4. Year 2. Develop and implement full bi-directional sequencing. This primarily
involves protocol application and software confirmation that the 8-dye transform
matrix works. Again, given the 2-laser, 2-region capabilities we have in version
1.0, this is completely feasible with current state of technology.

4.2 Electrophoresis System (CuraGen, SBio, Cornell)

4.2.0 System Design - Reusable (v. 1.1) to Disposable (v. 2.0) Separation
System

The 2+ years of production experience with the thin gel system at CuraGen,
combined with MIT's experience in the logistics of executing thousands of
electrophoresis runs/day, suggested to us separating the electrophoresis plates
and media from an electrophoresis station residing in an optical detection
compartment. Such a decoupling can reduce the optical "downtime" between runs to
minutes, thereby maximizing the throughput of the system. We propose to achieve
this efficient operation through a modular design:

1. an electrophoresis station integrating loading and electrophoresis
electronics with high speed thermal regulation;

2. female end connectors that contain the buffer, loading and optical
illumination accessories; and

3. gel plate devices (both thin slab and microchannel) that are either
disposable or remotely filled/cleaned.

These developments are readily tested on CuraGen's version 1.0 platform.

4.2.1 Electrophoresis Station/Accessories

CuraGen's version 1.0 thin slab device is a component that enables high voltage
electrophoresis with a variety of cooling (air, water, passive) and laser
illumination strategies. To reduce turnaround times (currently 20 minutes) and
to enable temperature sensitive gels and disposable gel devices, we will produce
a platform that accommodates a plate between two end-caps. A principle feature
is the decoupling of the electrophoresis system from the thermal regulation in a
safe, well-interlocked system, allowing direct and flexible testing of

[Confidential Treatment Requested]

Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------
microreplicated systems. Buffer caps have been designed that seal to the
radiused surface of the glass or plastic electrophoresis plates. For
non-disposable glass substrates, a separate pouring station is constructed to
enable rapid end-capping, pouring, and polymerization. Once prepared, the gel
plates are placed onto the electrophoresis station. Electrical contact is made
through the end-caps to the station, and final voltage contact is established
when the station lid is shut. Should longer lengths or better thermal control be
required, this system will be adaptable to different shapes and cooling
strategies. Our yearly plans are as follows:

Year 1. Evaluate materials and designs for the end-caps. Key considerations
include sealing to radiused glass/plastic gel plates, tensioning to the loading
combs, and proper tensioning of the plates. Prototypes will be machined from
delrin with non-conducting O-rings providing the sealing. We expect to address
first the sealing/loading characteristics, maintaining as much similarity to
working systems at CuraGen and MIT.

Year 2. Issues associated with producing large numbers of these systems,
including casting/molding, will be addressed in Years 2 and 3. Materials for
microreplicated gels substrates will be evaluated to insure their utility.

[GRAPHIC APPEARS HERE]

Figure 4.1 Electrophoresis system in version 2.0.

4.2.2 Microreplication Development

Our objective is to develop disposable microchannel substrates that can be
pre-filled with gel and supplied ready-to-use at low cost (less than $20/gel).
To accomplish this, we will investigate the production of lanes in plastic,
which has several advantages over a glass substrate: (1) lower production costs;
(2) reduced disposal and handling problems related to wet etch chemicals; (3)
facile bonding of base and cover substrates to form closed channels; and (4)
positionally-defined channel surface properties. These advantages are magnified
by the large consumption of plates, 500 to 1000 per day, in a high throughput
lab.

It is important to note that this effort is backed by experience with slab gels
(CuraGen), plastics and replication (Soane Technologies, Soane BioSciences), and
microfabricated electrophoresis channels (CuraGen with Cornell NNF). Rather than
a device on a glass microscope slide scale, our intent is to carry out
electrophoresis over suitable distances (10 cm - 30 cm) in a microchannel array
that takes advantage of proven loading and detection methodologies. In short, we
aim to replace the thin-gel plates we use now (10 cm and 14 cm width by 10 cm
and 30 cm length) with micro channel plates of the same size, increasing the
channel density to allow 64 (v. 1.1), 96 (v. 2.0), and ultimately 384 lanes.

Microreplication development will involve 6 component efforts: (a) establish
design features; (b) develop prototyping methods to test system designs and
plastic formulations; (c) investigate electrode deposition; (d) develop polymer
formulations exhibiting the required bulk properties; (e) develop channel
surface modifications to enable high-resolution separations; and (f) demonstrate
a microreplication production process. These six efforts, involving
contributions from CuraGen, Cornell and Soane, are reviewed below.

Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
---------------
-------------------------------------------------------------------------------

4.2.2.a Design Considerations
-----------------------------

The design considerations focus on developing a standard geometry for
interfacing the sample handling system, either combs or reactors, with the
sample imaging system (existing and proposed microfabricated optics discussed
below). Open lanes at the entrance converge in the imaging region in order to
present the correct size scale to each module. This strategy does not introduce
serpentine patterns that may broaden electrophoresis bands. We have already
fabricated masks to make converging lanes and will continue to investigate other
lane geometries, such as a fan of straight lanes at different angles. The
underlying principle is to have a size scale near the loading end that is
consistent with available solid phase and micromachined sample loading methods
and which will be amenable to microfabricated reactor arrays. [XXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]

4.2.2.b Substrate Prototyping
------------------------------

Prototyping methods are being established now and during the first year to lay a
foundation for testing different system designs (lane geometries, electrode
locations), polymer formulations, and surface modifications. We have identified
three methods for the prototyping of lanes. Most can be configured to
accommodate the long substrates required for electrophoresis.

[XXXXXXXXXXXXXXXXXXXXX]

[XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXX]

[Confidential Treatment Requested]

Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

[XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]

[XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]

4.2.2.d Formulation Development
-------------------------------

We will develop polymer formulations that are amenable to the processing
techniques outlined above, and which provide optical, mechanical, thermal and
surface characteristics to enable high-resolution separations and high-
sensitivity detection. We note that plastics are commonly used as an alternative
to glass in slab and tube gel electrophoresis, particularly for pre-cast gels
where cost is a concern. Plastic has also demonstrated utility in capillary
electrophoresis (e.g., Lukas and Jorgenson, 1985; Nielen, 1993; Liu et al.,
1993). The dominant use of fused silica in CE is due to the commercial
availability of high-quality fused silica capillaries, the higher thermal
conductivity and higher UV transparency of fused silica compared to plastic, and
the established means for modifying silica surfaces in CE separations. However,
none of these are inherent limitations in our system, and manufacturability is
actually a significant advantage of plastic in our approach.

Our focus will be on curable (UV, heat) and injection moldable materials, which
will be amenable to prototyping methods 2 and 3, and ultimately to production
methods (Section 4.2.2.f). Acrylics and aliphatic urethane acrylates will be
investigated first for their excellent optical properties and formulation
latitude. In general, they are highly transparent above 250nm and exhibit low
background fluorescence. Further, we have considerable experience in formulating
and processing these classes of polymers for optical applications. Any specific
formulations under consideration will be examined for transparency and
fluorescence before being used. Raw material purity is critical to eliminating
fluorescence, and we will implement established purification methods developed
by Soane Technologies. Mechanical rigidity can be imparted through crosslinking
or providing a rigid support (e.g., glass) under the microreplicated substrate.
The glass-plastic composite approach can also be used to improve thermal
dissipation if required, wherein the plastic layer need only be thicker than the
channel depths. Surface properties are considered separately in the next
section. Regarding potential scattering of the excitation beam, we view this as
a system issue and have designed channel geometries where the laser does not
pass through the channel walls.

4.2.2.e Surface Modification
----------------------------

[Confidential Treatment Requested]

Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

Surface chemistry and topology are critical in narrow bore separations, and
unmodified fused silica capillaries have traditionally required modification to
suppress electroosmotic flow in DNA sequencing applications. The two common
approaches are: (1) covalently linking the separation matrix to the capillary
wall, and (2) covalently or physically coating the capillary wall with a
hydrophilic polymer to suppress the surface charges and adsorption. Both in-situ
polymerized gels and replaceable matrices can be used with coated capillaries.
Like fused silica, most native polymer surfaces also exhibit a surface charge in
electrolyte solutions (Schutzner and Kenndler, 1992), and we will need to employ
one of the two basic strategies noted above. The silane coupling chemistry used
with fused silica is not applicable to organic polymer surfaces. However, there
are numerous methods for derivatizing polymer surfaces, and we have identified
three approaches to be investigated here as discussed below.

1. Surface Activation: Free radicals created at the polymer surface and present
---------------------
during acrylamide polymerization will result in covalent attachment between the
polyacrylamide and the surface. Liu et al. (1994) demonstrated a
hydroperoxidation preactivation method for covalently coating polypropylene
capillaries with polyacrylamide. This hydroperoxidation method should be
applicable to a wide variety of polymer surfaces, and will be used here. We can
also use electron discharges (RF, microwave, corona) in inert gas plasma to
activate the channel surfaces (Yasuda et al., 1987).

2. Coupling agents: This approach, analogous to silane coupling with silica,
------------------
involves a hetero bifunctional reagent, wherein one functional group reacts with
the polymer surface, and the other functional group (a vinyl or an acryl group)
later reacts with a growing polyacrylamide chain (e.g., urethane acrylates could
be used with surface hydroxyl groups). The first step is to ensure functional
groups are present at the polymer surface. We will design UV-curable
formulations containing latent functional groups that orient preferentially at
the surface (e.g., amine, carboxylic acid, hydroxyl groups). Other approaches to
incorporate surface functionality include using oxygen plasmas, and
photopolymerization of vinyl polymers (e.g. hydroxyethyl methacrylate) onto the
substrate surfaces (Wright, 1978). A wide range of hetero-bifunctional reagents
can then be used to react with these surface groups, and provide a reactive
group for incorporation into a polyacrylamide chain.

3. Surface Interpenetrating networks (SIPN's): This third approach is practiced
---------------------------------------------
by the contact lens industry (Thakrar and Gandhi, 1995). The polymer substrate
is first contacted with a liquid monomer (e.g., dimethyl acrylamide) which
penetrates the substrate surface without causing dimensional deformation. Next,
the gel-forming monomer solution (e.g., acrylamide) is injected into the channel
and polymerized. The first, surface-penetrating monomer will polymerize in the
presence of the free radicals, interpenetrating with the polymer substrate and
the gel matrix, forming a SIPN.

The above three methods provide us with a means for addressing a wide variety of
polymer substrates as we move through prototyping to production. The surface
activation method will be developed and applied for substrates fabricated by
direct patterning. The coupling agent approach will be developed in concert with
UV curable polymer formulations used for in-situ curing and replication.
Finally, we will explore surface activation, coupling agents and the SIPN
approach for applicability to molded thermoplastics. As described above, both
surface linkage of the gel matrix and coating of the channel surface are
feasible strategies, and either can be pursued using the three surface
modification methods. We will initially form polyacrylamide coatings on the
channels to enable quantitative evaluation of the surface treatment. This will
be done by measuring the EOF before and after surface coating. This enables
comparison of different polymer formulations and surface treatment methods. Upon
determining that a polymer composition and surface modification method are
acceptable, either surface coating followed by gel formation, or direct gel
formation with surface attachment can be used for evaluating separation
performance. While reference is made to polyacrylamide in this section, methods
applicable to polyacrylamide will be equally applicable to our advanced media
(Section 4.3).

4.2.2.f Development of Production Technology
---------------------------------------------
Casting or molding from a master substrate (microreplication) is expected to be
the most economical and robust production approach. The prototyping studies will
provide initial process parameters for replication using the optimized
formulation. A major technical difference between production and prototyping
will be the molds employed. For production, electroformed metal molds will be
preferred for ruggedness and economics. One method of producing electroformed
molds for micro scale parts is the well-known LIGA process depicted in Figure
4.3 (Becker et al., 1986). In this process, a thick (~100 (mu)m)
polymethylmethacrylate (PMMA) film is exposed to X-rays through an X-ray mask
which defines the lane geometries. The exposed areas are chemically altered and
can be selectively dissolved to form trenches. Nickel is electroplated into
these trenches to form a metal mold and the mold is released by dissolving the
remaining PMMA. This technology is well-established, but can prove costly due to
the need for access to a synchrotron for X-ray exposure and the expense of
fabricating

Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
-------------------------------------------------------------------------------
X-ray masks. Typically, LIGA is used to produce thick structures with fine
resolution (less than 1 (mu)m). In our application, this fine resolution is not
required and therefore we can use an alternate technology to manufacture metal
molds more cheaply. A photopatternable polyimide can be used in place of the
PMMA employed in the LIGA process. This allows us to use standard UV light
photolithographic tools and masks to form trenches for nickel electroplating.
This greatly reduces costs and a resolution of 10 microns can be easily achieved
in 100 micron thick structures.

[GRAPHIC APPEARS HERE]

Figure 4.3 Schematic representation of the LIGA processing for replicating
channel features.

As indicated earlier, microreplication can be accomplished by injection molding
or casting. The decision on replication method will be made in the third year
based on knowledge gained during the project, specifically on the processing
requirements of the optimized formulation and the replication accuracy
attainable with molding vs. casting. The objective in year three will be to
demonstrate on a small scale the production of high-quality microreplicated
systems from electroformed metal molds using the optimized polymer formulation.

4.3 Separation Media (SBio)

4.3.1 Reusable Media - v. 1.1

A reusable matrix will be developed to improve the efficiency of the Phase I
system in a production environment. Reuse of polyacrylamide has been
investigated in capillary and slab gel formats (Swerdlow et al., 1992 and 1994;
Ansorge et al., 1992). Three primary factors are reported to limit gel reuse:
(1) hydrolysis of amide bonds in polyacrylamide, (2) ion depletion at the
gel-liquid interface, and (3) difficult removal of DNA templates and large
fragments from the gel. As discussed in Section 3.3, SBio has developed
non-crosslinked matrices based on N-substituted acrylamides which provide high
resolution and greatly enhanced hydrolytic stability. We will develop
crosslinked gel formulations based on these hydrolytically-stable compositions
for use in the v. 1.1 system. We will also investigate the use of buffer
additives (Swerdlow et al., 1994) to match the environment of the gel and
running buffer, limiting the formation of a liquid junction potential. Rather
than monomers, uncrosslinked polymer solutions will be used as additives,
thereby limiting user exposure to toxic chemicals. Formamide and mixed
formamide/urea buffers will be investigated if standard 7M urea gels demonstrate
unacceptable deformation in the loading region (Swerdlow et al., 1994).

Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

[GRAPHIC APPEARS HERE]

Figure 4.4 A schematic diagram of the "collapsible" gel pore concept. The
framework of the polymer network will be formed by crosslinked,
hydrophilic, non-temperature-sensitive polymers (shown as thick
lines). Grafted onto the backbones of the crosslinked polymers will
be long, temperature-sensitive side-chains (thin lines). At one
temperature (A), the side-chains are more soluble and assume
extended conformations, "fingering" across the gel pore to form a
physically entangled network for the separation of DNA sequencing
fragments. When the temperature is changed (B), the side-chain
solubility is dramatically reduced, resulting in side-chain
collapse and dilation of the gel pores. The phase transition
temperature and the direction of the transition (low-to-high or
high-to-low temperature) can be controlled through the chemical
composition of the side-chains.

The above efforts should enable several runs per gel for samples not containing
template or large fragments. When these larger fragments are present, they may
remain in the gel or migrate out very slowly. We will investigate a "collapsible
pore" concept for dynamically opening the gel pores after a run to flush out
rapidly templates and large fragments. This scheme, depicted in Figure 4.4,
involves temperature-responsive side chains grafted onto a large-pore framework
of a non-temperature sensitive polymer. SBio has initial candidate compositions
for both the polymer framework and side chains from previous work, both of which
are hydrolytically stable. This research will involve synthesizing the graft
structures and testing different framework pore sizes and side chain molecular
weights. A variety of methods for polymerizing graft structures are known
(Dreyfuss and Quirk, 1987) and should be directly applicable here. A variation
of this strategy which does not require grafting is to synthesize the framework
polymer in an uncrosslinked solution of the temperature-responsive polymer. In
this approach, the temperature-sensitive polymer will be physically entrapped
within the framework structure, and will exhibit a transition from expanded to
collapsed structures (Section 3.3). Temperature-responsive polymers can be
designed to collapse due to either heating or cooling, providing additional
design flexibility.

4.3.2 Dynamic Porosity Media

The importance of long reads in genome sequencing is well established. In manual
sequencing, where the entire gel is imaged after a run, gradient gels have been
used to extend read length (States et al., 1991). However, the factors limiting
resolution and read length in manual and automated sequencing are different. As
reported by Slater and Drouin (1992), read length in a manual sequencer is
limited by the interband spacing of the large fragments, a problem which
directly lends itself to the gradient gel approach. In contrast, the read length
in automated slab sequencers is reported to be diffusion (band width) limited,
indicating that a static spatial gradient would not improve read length in
automated sequencers. Instead, longer gels and lower voltage drops have been
used to extend read lengths in automated sequencers (Luckey and Smith, 1993).

Theoretical and experimental evidence (Harke et al., 1992) indicates that the
resolution for a given DNA fragment length is optimized at a single gel
composition (other factors constant), and that this optimal gel concentration
decreases with increasing fragment length. Based on this evidence, we propose a
new strategy for increasing read length in automated sequencers. This strategy,
if successful, will be implemented in our v. 1.1 and v. 2.0 systems. We will
employ a temporal concentration gradient, reducing the effective gel
concentration during the run. The collapsible pore concept provides a basis for
developing dynamic porosity gels. We will design

Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

polymer side chains which convert from extended to collapsed conformation
gradually over a 10-20 degree range, creating a dynamic concentration gradient
in the pores via thermal control. Variations of this strategy include combining
different side chain compositions that have sharp transitions spanning a 10-20
degree range, and combining temperature-sensitive and non temperature-sensitive
side chains. We will investigate these different approaches and determine which
provides the optimal increase in read length. As in section 4.3.1, an alternate
approach to the grafted side chains will be temperature-sensitive uncrosslinked
polymer physically entrapped in a crosslinked framework. Dynamic porosity can,
of course, be coupled with other methods for increasing read length (e.g.,
longer gels) if necessary.

4.3.3 Media for Disposable Separation System - v. 2.0

We will develop media compositions and polymerization methods enabling the
microreplicated subsystem to be manufactured with self-contained media and
supplied ready-to-use. In production, the media will be polymerized in the
channels, followed by sealing the microreplicated system in a foil with excess
buffer (similar to how pre-cast protein gels are currently packaged).
Alternatively, the media can be supplied dehydrated in the channels, and then
rehydrated with buffer at the point of use. The gel-filled microreplicated
subsystem will need a shelf life of 6 months or greater. The matrix polymer
stability should not be a problem, as we can use the hydrolytically stable
polymers discussed in Section 4.3.1 if required. Urea is known to be unstable in
solution, however urea sequencing buffer is sold commercially with a reported
shelf life of over 6 months when refrigerated. We will determine whether urea
can if fact meet the shelf life requirements, or whether alternate denaturants
(Smith et al., 1990) need to be investigated. If these approaches are
unsuccessful, we can dehydrate the matrix in the channels for shipment. A
re-sealable cover/base design will be preferred in this approach for rehydration
kinetics, and should be feasible through the formulation of the cover material
(e.g., use of an elastomeric polymer layer enabling sealing through
compression). Polymerization of crosslinked gels in capillary size channels can
result in shrinkage-induced defects, particularly when the gel is linked to the
channel walls. SBio's patented process (Soane, 1991) with proven ability to
prepare defect-free, gel-filled capillaries can be employed here. This method
compensates for polymerization-induced shrinkage by a sequential curing
technique similar to that described for substrate replication. This process can
be readily applied to all of the channels simultaneously using a single UV scan
from one end to the other of the sealed microreplicated system. The above
methods should also allow for incorporation of dynamic porosity compositions for
situations where longer read lengths are desired.

4.4 Advanced Detection: Replicated Optics (Cornell NNF, CuraGen)

We are proposing to fabricate and test a microfabricated optical system for
excitation and detection of fluorescence from channels. This optical system is
designed to dovetail into the microreplicated gel systems. The system employs
arrays of micro[XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]

4.4.1. Side Illumination Spectral Detection.

Building on CuraGen's grating spectrograph, we will extend the techniques of
micro-optics to make a 10-fold reduction in the size and cost of the v. 1.0
imaging system. The 0.5 numerical aperture described above implies a large
collection angle. This is desirable for collecting the maximum amount of light
and results in a very short depth of focus. This will result in the compact
system we desire and will allow a dense array of optical elements and efficient
light collection. High quality lens arrays are commercially available in this
range of parameters, e.g. Gelsil Inc., which uses the Corning technology for
high quality molded silica optics. In the final phase of the work after
optimizing the design and testing prototype systems, our efforts will be
directed toward reducing the number of optical elements by combining
functionalities. For example, the wavelength dispersion and spatial focusing of
the fluorescent radiation can be combined in one unit by forming a grating on
the surface of the lens array. While our schematic shows discrete optical
elements, we will work to combine these into a maximally integrated unit for
increased simplicity in and operation.

[Confidential Treatment Requested]

Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

[XXXXXXXXXXXXXXXXXXX]

Figure 4.4 Schematic of microfabricated optical elements that accomplish the
same function as CuraGen's existing device at a 10-fold higher
throughput. For side laser illumination, the confocal laser and
dichroic can be omitted.

4.4.2. Confocal Illumination and Detection.

The fluorescence collection system we have outlined will work with a variety of
laser illumination methods. Depending on the outcome of the channel prototyping,
we may chose to explore the possibilities of confocal illumination. The
outstanding feature, compared to the conventional scanning confocal fluorescence
method, is that we do not need to slew mechanically the optics across the
capillary array. The result is a more robust design. We can also employ rigid
alignment methods, impossible in a scanned system, required for the short depth
of focus of the high numerical aperture lens system.

4.4.3. Laser illumination

It is intended to investigate the use of both pulsed and continuous wave laser
source excitation with the objective of ascertaining the minimum limits of
fluorophore concentration for the reliable identification of fluorophore tags.
Of particular importance is the level of unwanted background light generated by
Rayleigh and Raman scattering in the gel and surrounding media, specular
reflection of the laser beam into the collection optics and any fluorescent
emission produced in the glass components of the system by the laser. We will
investigate the tradeoffs involved with continuous wave excitation (with and
without scanning) and pulsed laser excitation with

[Confidential Treatment Requested]

Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

respect to signal-to-noise ratio and observation time and ascertain minimum
fluorophore detection limits under these conditions.

4.4.4. Production.

Once the lane and optical features have been resolved, we will complete
development by characterizing methods for producing these in large quantities.
In particular, we will explore replication means similar to those being
evaluated for the formation of channel features. Principle difficulties will be
to develop robust means for replicating sub-micron features. However, previous
research in fabricating micro-optics has show that such resolution is
achievable.

4.5 Informatics (CuraGen,MIT)

4.5.1 Image Analysis and Base Calling

The preprocessing module will be modified to allow the use of more than 4 dyes
with the base calling software. For the 5-dye system, this can be accomplished
simply by adding a spectrally distinct 5th dye and using a 5x5 matrix for the
transformation from "wavelength space" to "dye space." The conversion produces 5
independent signal channels, which can be used in any base-coding scheme. For
example, 4 channels can be used to identify bases in one DNA strand, while the
5th channel identifies a single base in the reverse strand as an aid in sequence
alignment. For the 8-dye system, one option is as follows. Two lasers are used
to excite two spectrally distinct sets of four dyes each, each in two
independent wavelength regions. Thus the same 4x4 matrix operations used in
conventional preprocessing can be applied independently to the sets of four
dyes. The combined wavelength transform is then a block-diagonal 8x8 matrix with
two 4x4 blocks. We will investigate the possibility of using an analogous matrix
transform to deconvolute the signal from many dyes directly, even if there is
some spectral overlap between dyes.

The base calling module will be redesigned to produce a statistical measure of
the likelihood that a particular signal was generated by each of the four
nucleotides. This probability function can be obtained, for instance, by
comparing the measured signal to the expected signal, or "cluster center," for
each of the four bases (see Figure 3.11). For instance, one can define a metric
which measures the distance between the experimental signal and each of the four
cluster centers. If one of these distances is much smaller than the other three,
then the identity of the base is clear. We will be conducting ongoing research
to investigate the optimal form for the distance metric, including such factors
as the length of the DNA sequence and the identities of nearby bases. We will
also investigate different algorithms for converting distances from cluster
centers into base call probabilities. The best form will be investigated in
conjunction with our ongoing research into the best method of "training," i.e.
the optimum approach to allocating and locating base call prototypes within each
cluster.

4.5.2 Assembly and Finishing

While various definitions of finished sequence have been proposed, they are
usually based on a specific level of accuracy (e.g. 1 error in 1000 bases). The
direct incorporation of the complete information on a base call will allow for
full information to be taken into account during assembly, as opposed to an
arbitrary single call. For an assembled sequence, this translates to a
quantifiable confidence level that can be experimentally correlated with an
error rate. Integration of the base call information with the assembled sequence
will allow the contiguous sequence to be scanned and the quantitative confidence
in the finished sequence to be displayed.

A suitable function of the distances to base call clusters described above and
shown in Figure 3.10 can be used in multiple alignments to greatly increase the
confidence in finished sequence. The goal is demonstrated with a simple example,
showing only two bases. If a base is sequenced three times as A, A, and T, it is
difficult to determine a consensus with confidence. However if the same base is
sequenced as (A=0.95,T=0.05), (A=0.98,T=0.02), and (T=0.59,A=0.41), then it is
reasonable to declare with confidence that A is the correct call. In practice,
we will use the information available to create statistical measures of the
likelihood that each base call is correct, and then will use these to generate
mismatch penalty tables to be used in existing multiple alignment tools.
Furthermore, we will investigate other options for more direct conflict
resolution in multiple alignments. These will use the full information available
from the base calling software.

Our approach will lead to an integration of the base calling software directly
into the assembly and finishing process. This tie-in will allow for sequence
assembly and finishing to take place in a statistically valid and fully
automated fashion.

Automated Sequencing System for the Human Genome Project
RFA: HG-95-004

Collaborators Principal Investigators
------------- -----------------------

Cornell University Dr. Harold G. Craighead

Soane BioSciences, Inc. Dr. Herbert H. Hooper

The Whitehead Institute for Biomedical Research Dr. Trevor L. Hawkins, Ph.D.

(See attached confirming letters)

Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

8. Consortium/Contractual Arrangements

Automated Sequencing System for the Human Genome Project

RFA: HG-95-004

CuraGen Corporation, Cornell University, Soane BioSciences, Inc. & The Whitehead
Institute for Biomedical Research hereby state that there are currently in place
the necessary programmatic, fiscal and administrative arrangements to ensure
that all necessary legal, financial and administrative matters are dealt with in
the appropriate manner.

In addition, CuraGen Corporation hereby confirms (see attached letters received
from each of the respective consortia) that all of the required programmatic and
administrative personnel of all consorita involved are aware of the NIH
consortium grant policy and are prepared to establish the necessary
interorganization agreements that will ensure compliance with all Federal
regulations and policies.

Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

Appendix

PI: Gregory T. Went

Project Title: Automated Sequencing System for the Human Genome Project

Appendix 1 - Color Figures

Appendix 2- Detailed Cost Analysis

Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------

9. References

Ansorge, W., et. al., High-throughput automated DNA sequencing facility with
fluorescent labels at the European Molecular Biology Laboratory,
Electrophoresis 13: 616 (1992).
Babu, Y. et. al., Preparation of Polyacrylamide Gel Filled Capillaries for
Ultra-high Resolution of Polynucleotides by Capillary Gel Electrophoresis.
Anal. Chem. 64:1221 (1992).
Balch et. al., Sequencing of DNA in Micromachined Channels, presented at Genome
Sequencing and Analysis Conference, Hilton Head, Sept. 17-21, 1994.
Bare, H. Haas, F. Honey,D. Mikolas, D. Craighead, H. A simple surface emitting
LED array for free-space optical interconnects. Photonics Technol. Lett.,
5:172 (1993).
Barker, D.L., High throughput mapping using capillary arrays, presented at The
Human Genome Project: Commercial Implications, San Francisco, March 6-8,
1995.
Bashkin, J., M. Bartosiewicz, D. Barker, and R. Johnston, High throughput DNA
analysis by capillary array electrophoresis, presented at the 17th
International Symposium on Capillary Chromatography and Electrophoresis,
Virginia, May 7-11, 1995.
Beck,S., and Alderton,R. A strategy for the amplification, purification, and
selection of M13 templates for large-scale DNA sequencing. Anal Biochem 212:
498 (1993).
Becker, et. al., Fabrication of microstructures with high aspect ratios and
great structural heights by synchrotron radiation lithography,
galvanoforming, and plastic molding (LIGA process), Microelectronic
Engineering 4:35 (1986).
Brumley, R.L. and L.M. Smith, Rapid DNA Sequencing by Horizontal Ultrathin Gel
Electrophoresis, Nucl. Acids Res. 19: 4121 (1991).
Busch, M.H.A., J.C. Kraak, and H. Poppe, Cellulose acetate-coated fused silica
capillaries for the separation of proteins by capillary zone
electrophoresis, J. Chrom. A. 695: 287 (1995).
Chang, E.I. and Lippmann, R.P. A boundary hunting radial basis function
classifier which allocates centers constructively, in Advances in neural
information processing systems 5, Hanson, et. al., eds. pp 139 (1993).
Chen, F. and Craighead H. Diffrative Phase Elements Based on Two-Dimensional
Artificial Dielectrics. Optics Lett. 20:121 (1995).
Chin, A.M., and J.C. Colburn, Counter-migration capillary Electrophoresis in DNA
Restriction Fragment Analysis, Am. Biotech. Lab. 7: 16 (1989).
Dreyfuss, P., and R.P. Quirk, Graft Copolymers, in Encyclopedia of Polymer
Science and Engineering, 2nd Edition, John Wiley and Sons, (1987).
Effenhauser, C.S. et. al., Glass Chips for High-Speed Capillary Electrophoresis
Separations with Submicrometer Plate Heights, Anal. Chem. 65:2637 (1993).
Effenhauser, C.S., et. al., High-Speed Separation of Antisense Oligonucleotides
on a Micromachined Capillary Electrophoresis Device, Anal. Chem. 66: 2949
(1994).
Epperson and Denton, Binning Spectral Images in a Charged COupled Device.
Anal.Chem. 61: 1513 (1989).
Fleischmann et. al., (1995) Science 269: 496
Fry,G., et. al., A new approach to template purification for sequening
applications using paramagnetic particles. BioTechniques 13: 124 (1992).
Giddings, M.C., et. al., An adaptive object oriented strategy for base calling
in DNA sequence analysis. Nucleic Acids Research 21: 4540 (1993).
Gilges, M., et. al., Capillary Zone Electrophoresis Separations of Basic and
Acidic Proteins using Poly(vinyl alcohol) Coatings in Fused Silica
Capillaries, Anal. Chem. 66: 2038 (1994).
Golden III, J.B. et. al., Pattern recognition for automated DNA sequencing: I.
On-line signal conditioning and feature extraction for base calling, in
Proceedings of the first international conference on intelligent systems for
molecular biology, Hunter et. al., eds. AAAI Press, Menlo Park, CA (1993).
Grossman, P.D., Electrophoretic separation of DNA sequencing extension products
using low-viscosity entangled polymer networks, J. Chrom. A. 663: 219
(1994).
Grossman, P.D., and D.S. Soane, Capillary Electrophoresis of DNA in Entangled
Polymer Solutions, J. Chrom. 559: 257 (1991).
Harke, H.R., S. Bay, J.Z. Zhang, M.J. Rocheleau and N. Dovichi, Effect of Total
Percent Polyacrylamide in Capillary Gel Electrophoresis for DNA Sequencing
of Short Fragments, J. Chrom. 608: 143 (1992).
Harrison, D. et. al., Micromachining a Miniaturized capillary Electrophoreisi-
based chemical Analysis System on a Chip. Science 261: 895 (1993).
Harrison, D.J., A. Manz, Z. Fan, H. Ludi, and H.M. Widmer, Capillary
Electrophoresis and Sample Injection Systems Integrated on a Planar Glass
Chip, Anal. Chem. 64: 1926 (1992b).
Harrison, D.J., et. al., Micromachining a miniature capillary electrophoresis-
based chemical analysis system on a chip, Science 261: 895 (1992).

Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------
Harrison, et. al., Capillary Electrophoresis and sample Injection Systems
Integrated on a Planar Glass Chip. Anal. Chem. 64: 1926 (1992).
Hawkins,T.L. M13 single-stranded purification using a biotinylated probe and
streptavidin coated magnetic beads. DNA sequence-J.DNA Sequencing and
Mapping. 3. 65 (1994).
Hawkins,T.L. The use of magnetic particles in molecular biology. In Automated
DNA sequencing and analysis techniques. Ed. Craig Venter,J. Academic Press
(1994).
Hawkins,T.L., et. al., DNA purification and isolation using a solid-phase.
Nucl.Acids.Res. 22: 4543 (1992).
Hawkins,T.L., et. al., Solid-phase molecular biology. Biomedical
products.December issue . Ed Steve Ernst (1994).
Heiger, D.N., et. al., Separation of DNA Restriction Fragments by High
Performance Capillary Electrophoresis with Low and Zero Crosslinked
Polyacrylamide using Continuous and Pulsed Electric Fields, J. Chrom. 516:
33 (1990).
Huang, X.C., et. Al., DNA Sequencing using Capillary Array Electrophoresis,
Anal. Chem. 64: 2149 (1992).
Huang, X.C., et. al., Capillary Array Electrophoresis Using Laser-Excited
Confocal Fluorescence Detection, Anal. Chem. 64: 967 (1992a).
Hultman, T et. al., Bi-directional solid-phase sequencing of In Vitro-amplified
plasmid DNA. BioTechniques 10: 84 (1991).
Hultman,T., et. al., Direct solid-phase sequencing of genomic and plasmid DNA
using magnetic beads as solid support. Nucl.Acids.Res. 17: 4937 (1989).
Jacobson, S.C., et. al., Microchip Capillary Electrophoresis with an Integrated
Postcolumn Reactor, Anal. Chem. 66: 3472 (1994).
Jacobson, S.C., et. al., Effects of Injection Schemes and Column Geometry on the
Performance of Microchip Electrophoresis Devices, Anal. Chem. 66: 1107
(1994).
Jacobson, S.C., et. al., Precolumn Reactions with Electrophoretic Analysis
Integrated on a Microchip, Anal. Chem. 66: 4127 (1994).
Johnson, B, et. al., The design of separation media for DNA sequencing in
capillaries, poster at the Human Genome Project: Commercial Implications,
San Francisco, March 6-8, 1995.
Kostichka, et al., High Speed automated DNA sequencing in ultrathin gels.
BioTechnology, 10, 78-81. (1992).
Kovacs, G.T.A., and K.O. Holland, Electrophoretic System, European patent
application 89313379.3 (1989).
Lander, et al.. Personal communication, Pilot study to sequence the human genome
(1995)
Liu, P.Z., et. al., Polyacrylamide-Modified Polypropylene Hollow Fibers for
Capillary Electrophoresis, J. Microcolumn Sep. 6: 581 (1994).
Liu, P.Z., et. al., Polymeric hollow fibers for capillary electrophoresis, J.
Microcolumn Sep. 5: 245 (1993).
Luckey, J.A., and L.M. Smith, Optimization of the Electric Field Strength for
DNA Sequencing in Capillary Gel Electrophoresis, Anal. Chem. 66: 2841
(1993).
Lukacs, K.D., and J.W. Jorgenson, Capillary Zone Electrophoresis: Effect of
Physical Parameters on Separation Efficiency and Quantitation, J. High
Resol. Chrom. 8: 407 (1985).
Manz, A., et. al., Miniaturization of Chemical Analysis Systems - A Look into
Next Century's Technology or Just a Fashionable Craze?, Chimia 45: 103
(1991).
Mathies and Huang, Nature 359: 176 (1992).
Mikolas, D. et. al., Fabrication of Aspheric High Numerical Aperture Reflective
Diffractive Optic Elements Using Electron Beam Lithography. J. Vac. Sci.
Technol. B 12: 20 (1994).
Nielen, M.W., Capillary zone electrophoresis using a hollow polypropylene fiber,
J. High Resolut. Chrom. 16: 62 (1993).
Olejnik, J., et. al., Photocleavable biotin derivatives: A versatile approach
for the isolation of biomolecules. Proc. Natl. Acad. Sci. USA 92: 7590
(1995).
Pace, S.J., Silicon Semiconductor Wafer for Analyzing Micronic Biological
Samples, US Patent 4,908,112, Issued (1990).
Pulyaeva, H., et. al., Molecular Sieving of Lambda Phage DNA in Polyacrylamide
Solutions as a function of the Molecular Weight of the Polymer,
Electrophoresis 13: 608 (1992).
Righetti, P.G., et. al., Towards New Formulations for Polyacrylamide Matrices,
as Investigated by Capillary Zone Electrophoresis, J. Chrom. 638: 165
(1993).
Ruiz-Martinez, et. al., DNA Sequencing by Capillary Electrophoresis with
Replaceable Linear Polyacrylamide and Laser-Induced Fluorescence Detection,
Anal. Chem. 65: 2851 (1993).
Schutzner, W. and E. Kenndler, Electrophoresis in Synthetic Organic Polymer
Capillaries: Variation of Electroosmotic Velocity and (zeta) Potential with
pH and Solvent Composition, Anal. Chem, 64, (1991).
Shank, S. and Craighead, H. Binary Optics for Optical Interconnects, S. M. Shank
and H. G. Craighead. Proc. SPIE, 2216 (1994).
Shank, S. et. al., Multiple-level phase gratings fabricated using focused ion
beam milling and electron beam lithography. J. Vac. Sci. Technol B 12: 3643
(1994).

Principal Investigator/Program Director (Last, first, middle):
Went, Gregory T.
----------------
--------------------------------------------------------------------------------
Slater, G.W., and G. Drouin, Why can we not Sequence Thousands of DNA Bases on a
Polyacrylamide Gel, Electrophoresis 13: 574 (1992).
Smith et al., Fluorescent detection in automated DNA sequence analysis. Nature,
321, 674 (1986).
Smith, C.L., et. al., Rapid Sequencing of DNA, Am. Biotech Lab 8: 48 May (1990).
Soane, D.S., Replaceable Crosslinked Gels for DNA Analysis by Capillary
Electrophoresis, Third International Symposium on Capillary Electrophoresis,
York, UK, August 24-26, 1994.
Soane, D.S., Advances in Gel Matrices for DNA Electrophoresis, presented at The
Human Genome Project: Commercial Implications, San Francisco, March 6-8,
1995.
Soane, D.S., and Y.C. Bae, Separation Media for Electrophoresis, International
patent application WO 94/10561.
Soane, D.S., Controlled Casting of Shrinkable Material, US Patent 5,110,514
(1992).
Soane, D.S., Gel Casting Method and Apparatus, US Patent 5,061,336 (1991).
States, J.C., et. al., Electrophoresis System for Resolving Over 500 Nucleotides
with a Single Sample Loading, BioTechniques 46: (11), 1 (1991).
Sulston,J.,et. al., The C.elegans genome sequencing project: a beginning. Nature
356: 37 (1992).
Swerdlow, H., et. al., Reloading and Stability of Polyacrylamide Slab Gels for
Automated DNA Sequencing, BioTechniques 16: (4), 684 (1994).
Swerdlow, H., et. al., Stability of Capillary Gels for Automated Sequencing of
DNA, Electrophoresis, 13, 475 (1992).
Takahashi, et. al., Multiple Sheath-Flow Gel capillary-Array electrophoresis for
Multicolor Fluorescent DNA Detection. Anal. Chem. 66: 1021 (1994).
Taylor, J. and Yeung, E. Axial-Beam Laser-Excited Fluorescence Detection in
Capillary Electrophoresis. Anal. Chem. 64: 1741 (1992).
Terry, S.C., et. al., A Gas Chromatographic Air Analyzer Fabricated on a Silicon
Wafer, IEEE Transactions, 26: 1880 (1979).
Thakrar, A.R. and K. Gandhi, Method of Manufacturing a Contact Lens, US Patent
5,433,898 (1995).
Tibbetts, C.J., et. al., Neural networks for automated base calling of gel-based
DNA sequencing ladders, in Automated DNA sequencing and analysis techniques,
Adams xx.xx., eds. Academic Press, London (1994).
Ueno, K. and E.S. Yeung, Simultaneous Monitoring of DNA Fragments Separated by
Electrophoresis in a Multiplexed Array of 100 Capillaries, Anal. Chem. 66:
1424 (1994).
Uhlen, M, Magnetic separation of DNA. Nature 340: 733 (1989).
Watson,A., Smalldon,N., Lucke,R., and Hawkins,T.L., The Caenorhabditis elegans
project: first steps in automation. Nature 362: 569 (1993).
Went, G.T., Development of an Integrated Microfabricated Device for DNA Fragment
Analysis, Biochip Array Technologies Conference, Washington, DC, May 10,
1995.
Westphall et. al., Proc SPIE 2386: 45 (1995).
Wilson, R., et. al., 2.2Mb of contiguous nucleotide sequence from chromosome III
of C.elegans. Nature 368: 32 (1994).
Woolley, A.T., and R.A. Mathies, Ultra-High-Speed DNA Fragment Separations Using
Microfabricated Capillary Electrophoresis Chips, Proc. Natl. Acad. Sci. USA,
91: 11348 (1994).
Wright, A.N., Photopolymerization at Surfaces, in Polymer Surfaces, D.T. Clark
and W.J. Feast Eds., John Wiley and Sons, (1978).
Yasuda, H.K., et. al., Plasma-Surface Interactions in the Plasma Modification of
Polymer Surfaces, in Polymer Surfaces and Interfaces, W.J. Feast and H.S.
Munro Eds, John Wiley and Sons, (1987).
Zhu, M., et. al., Factors Affecting Free Zone Electrophoresis and Isoelectric
Focusing in Capillary Electrophoresis, J. Chrom. 480: 311 (1989).

II Principal Investigator Program Director (Last, first, middle):

--------------------------
--------------------------------------------------------------------------------
CHECKLIST
--------------------------------------------------------------------------------

TYPE OF APPLICATION

[X] NEW application. (This application is being submitted to the PHS for the
first time.)

[_] REVISION of application number.
------------------------------------------
This application replaces a prior unfunded version of a new, competing
continuation, or supplemental application.

[_] COMPETING CONTINUATION of grant number.
----------------------------------
(This application is to extend a funded grant beyond its current
project period.)

[_] SUPPLEMENT to grant number.
----------------------------------------------
(This application is for additional funds to supplement a currently funded
grant.)

[_] CHANGE of principal investigator program/program director.
Name of former principal investigator program ???????????.
-----------------

[_] FOREIGN application, city and country of birth and present citizenship of
principal investigator/program director. (This information is required by
the U.S. Department of State.)
---------------------------------------------
--------------------------------------------------------------------------------
1. ASSURANCES/CERTIFICATIONS

The following assurances/certifications are made by checking the appropriate
boxes and are verified by the signature of the OFFICIAL SIGNING FOR APPLICANT
ORGANIZATION on the FACE PAGE at the application. Descriptions of individual
assurances/certifications begin on page 24 at Specific Instructions.
--------------------------------------------------------------------------------

a. Human Subjects (Complete Item 4 on the Face Page)

[_] Full IRB Review [_] Expedited Review

b. Vertevrate Animals (Complete Item 5 on the Face Page)

c. and Patents (Competing Continuation Application Only-- Complete Item
10 on the Face Page)

d. Department and Suspension [X] No [_] Yes (Attach explanation)

e. Drug-Free Workplace (Applicable only to new or revised applications being
submitted to the PHS for the first proposed project. Type 1)

[X] Yes [_] No (Attach explanation)

f. Lobbying

With Federal appropriated funds [X] No

With other than Federal appropriated funds [X] No [_] Yes
(If "Yes." see page 29 and
attach Standard Form LLL
"Disclosure of Lobbying
Activities" to the application
behind the second page of the
Checklist)

g. Delinquent Federal Debt [X] No [_] Yes (Attach explanation)

h. Misconduct in Science (Form PHS 6315) [X] Fired [_] Not Fired
If filed, date of initial Assurance or Latest Annual Report 2/21/95
-----------------

i. Civil Rights j. Handicapped Individuals K. Sex Discrimination l. Age Discrimination
Form HHS 441 Form HHS 641 Form HHS 639-A Form HHS 680

[X] Filed [X] Filed [X] Filed [X] Filed

[_] Not Filed [_] Not Filed [_] Not Filed [_] Not Filed

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------
CHECKLIST (Continued)
--------------------------------------------------------------------------------
2. PROGRAM INCOME (See instructions, page ??)

All applicants must indicate (Yes or No) whether program income is anticipated
during the ???????) for which grant support is requested.
[X] No [ ] Yes If "Yes." use the format below to reflect the amount and
source(s) of anticipatedprogram income.

--------------------------------------------------------------------------------
?????? ????? Anticipated Amount Source(s)
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

3. INDIRECT COSTS

Indicate the applicant organization's most recent indirect cost rate established
with the appropriate DHHS Regional Office, or, in the case of for profit
organizations, the rate established with the appropriate PHS Agency Cost
Advisory Office. If the applicant organization is in the process of initially
developing or renegotiating a rate, or has established a rate with another
Federal agency, it should immediately upon notification that an award will be
made develop a tentative indirect cost rate proposal. This is to be based on its
most recently completed fiscal year in accordance with the principles set forth
in the pertinent DHHS Guide for Establishing Indirect Cost Rates and submitted
to the appropriate DHHS Regional Office or PHS Agency Cost Advisory Office.
Indirect costs will not be paid on foreign grants, construction grants, grants
to Federal organizations, and grants to individuals and usually not on
conference grants. Follow any additional instructions provided for Research
Career Development Awards. Institutional National Research Service Awards, and
the specialized grant applications listed on page 6.

[_] DHHS Agreement dated:______________ [_] No Indirect Cases Requested.

[_] DHHS Agreement being negotiated with _______________ Regional Office.

[X] No DHHS Agreement cut rate being negotiated with * Date April 24, 1995
CALCULATION*
(The entire grant application, including the Checklist will be ???????? and
provided to peer reviewers as CONFIDENTIAL ???????????. ???????? ??? following
information on interest costs if OPTIONAL for ??????? organizations.)

a. Initial budget period:
Amount of base [XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]

b. Entire proposed project:
Amount of base [XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX]

(1) Add to total direct costs from form page 4 and enter new total on FACE
Page. Item 7?.
(2) Add to total direct costs from form page 5 and enter new total on FACE
Page. Item 8?.

*Check appropriate box(es):

[_] Salary and wages case [X] Modified total direct costs base

[_] Other base (Explain below)

[_] Off-site, other special rate, or more than one rate involved (Explain below)

Explanation (Attach separate sheet, if necessary.):

* National Institute of Standards and Technologies
Atlanta, GA

[Confidential Treatment Requested]

KK Principal Investigator/Program Director (Last, first, middle)
Went, Gregory T.
----------------
--------------------------------------------------------------------------------
Attach this form to the signed original of the
application after the CHECKLIST. Do not duplicate.

PERSONAL DATA ON
PRINCIPAL INVESTIGATOR/PROGRAM DIRECTOR
--------------------------------------------------------------------------------

The Public Health Service has a continuing commitment to monitoring the
operation of its review and aware processes to detect--and deal
appropriately with--any instances of real or apparent inequities with
respect to age, sex, race or ethnicity of the proposed principal
investigator/program director.

To provide the PHS with the information it needs for this important task,
complete the form below and attach it to the signed original of the
application after the Checklist. Do not attach copies of this form to the
duplicated copies of the application.

Upon receipt and assignment of the application by the PHS, this form will
be separated from the application. This form will not be duplicated, and it
will not be a part of the review process. Data will be confidential and
will be maintained in Privacy Act record system 09-25-0036. "Grants:IMPAC
(Grant/Contract information)." All analysis conducted on the data will
report aggregate statistical findings only and will not identify
individuals.

If you decline to provide this information, it will no way affect
consideration of your application.

Your cooperation will be appreciated.

================================================================================
DATE OF BIRTH (month/day/year) SEX

July 1, 1963 [_] Female [X] Male

--------------------------------------------------------------------------------
PLACE AND/OR ETHNIC ORIGIN (check one)

Note: The category that most closely reflects the individual's recognition in
the community should be used for purposes of recording mixed racial and/or
ethnic origins.

[_] American Indian or Alaskan Native. A person having origins in any of the
original peoples of North America and who maintains a cultural
identification through tribal affiliation or community recognition.

[_] Asian or Pacific Islander. A person having origins in any of the original
peoples of the Far East. Southeast Asia, the indian subcontinent, or the
Pacific islands. This area includes, for example, China, India, Japan,
Korea, the Philippine Islands, and Samoa.

[_] Black, not of Hispanic origin. A person having origins in any of the black
racial groups of Africa.

[_] Hispanic. A person of Mexican, Puerto Rican, Cuban. Central or South
American, or other Spanish culture or origin, regardless of race.

[X] White, not of Hispanic origin. A person having origins in any of the
original peoples of Europe, North Africa, or the Middle East.

[_] Check here if you do not wish to provide some or all of the above
information.

Document Metadata

Table of Contents

Exhibit 10.15 CuraGen Corporation has omitted from this Exhibit 10.15 portions of the Agreement for which CuraGen Corporation has requested confidential treatment from the Securities and Exchange Commission. The portions of the Agreement for which...

Document Metadata