Determination of single-nucleotide polymorphisms by real-time pyrophosphate DNA sequencing

The characterization of naturally occurring variations in the human genome has evoked an immense interest during recent years. Variations known as bia llelic Single-Nucleotide Polymorphisms (SNPs) have become increasingly popu lar markers in molecular genetics because of their wide application both in evolutionary relationship studies and in the identification of susceptibil ity to common diseases. We have addressed the issue of SNP genotype determi nation by investigating variations within the Renin-Angiotensin-Aldosterone System (RAAS) using pyrosequencing, a real-time pyrophosphate detection te chnology. The method is based on indirect luminometric quantification of th e pyrophosphate that is released as a result of nucleotide incorporation on to an amplified template. The technical platform employed comprises a highl y automated sequencing instrument that allows the analysis of 96 samples wi thin 10 to 20 minutes. In addition to each studied polymorphic position, 5- 10 downstream bases were sequenced for acquisition of reference signals. Ev aluation of pyrogram data was accomplished by comparison of peak heights, w hich are proportional to the number of incorporated nucleotides. Analysis o f the pyrograms that resulted From alternate allelic configurations for eac h addressed SNP revealed a highly discriminating pattern. Homozygous sample s produced clear-cut single base peaks in the expected position, whereas he terozygous counterparts were characterized by distinct half-height peaks re presenting both allelic positions. Whenever any of the allelic bases of an SNP Formed a homopolymer with adjacent bases, the nonallelic signal was add ed to those of the SNP. This feature did not, however, influence SNP readab ility. Furthermore, the multibase reading capacity of the described system provides extensive flexibility in regard to the positioning of sequencing p rimers and allows the determination of several closely Located SNPs in a si ngle run.