5HTT rs25531 SNP Sample Clauses

5HTT rs25531 SNP. Genotyping of the rs25531 SNP, the G allele of which is almost always inherited with the L allele and reduces transcriptional activity to that of the S allele, was also attempted. The protocol proposed by Xxxxxxxx et al. (2006) was followed. Broadly it specifies a method similar to the above but with an additional digest step before electrophoresis (Xxxxxxxx, Xxxxxx et al. 2006). 200nM of forward (5'-TCCTCCGCTTTGGCGCCTCTTCC-3') and reverse (5'- TGGGGGTTGCAGGGGAGATCCTG-3') primers were included along with the reagents specified in the section above at the same concentrations and 5 ng of DNA in a total reaction volume of 20 µl. Following PCR cycling (95ºC for 15 minutes, followed by 35 cycles of (94ºC for 30 seconds, 65.5 ºC for 90 seconds, 72 ºC for 60 seconds), 72ºC for 10 minutes) 7 µl of product was digested with 1x buffer and HpaII (New England Biolabs) in a total volume of 20 µl for 3 hours. The G allele of rs25531 creates a restriction site recognised by HpaII which cuts the DNA fragment creating two bands, whereas fragments carrying the more common A allele are not digested. Lastly, 4 µl of the restriction digest and 18 µl of the original PCR product were loaded onto a 3% agarose gel. This assay was first tested on DNA from several different sources but the bands after digestion were too faint to make a reliable genotype call. In order to address this problem the assay was optimised by first removing Q solution and then by adding 0, 1, 2 and 3 µl of MgCl2, as suggested by the manufacturers (Qiagen). However in no instance did this improve the quality of the DNA bands visualised by electrophoresis. Due to time constraints at this stage of the project and the need to process a large number of samples a high throughput method of genotyping 5HTTLPR was opted for and the genotyping of rs25531 was postponed.