Determination of Single-Nucleotide Polymorphisms by Real-time Pyrophosphate DNA Sequencing

Abstract

The characterization of naturally occurring variations in the human genome has evoked an immense interest during recent years. Variations known as biallelic Single-Nucleotide Polymorphisms (SNPs) have become increasingly popular markers in molecular genetics because of their wide application both in evolutionary relationship studies and in the identification of susceptibility to common diseases. We have addressed the issue of SNP genotype determination by investigating variations within the Renin-Angiotensin-Aldosterone System (RAAS) using pyrosequencing, a real-time pyrophosphate detection technology. The method is based on indirect luminometric quantification of the pyrophosphate that is released as a result of nucleotide incorporation onto an amplified template. The technical platform employed comprises a highly automated sequencing instrument that allows the analysis of 96 samples within 10 to 20 minutes. In addition to each studied polymorphic position, 5-10 downstream bases were sequenced for acquisition of reference signals. Evaluation of pyrogram data was accomplished by comparison of peak heights, which are proportional to the number of incorporated nucleotides. Analysis of the pyrograms that resulted from alternate allelic configurations for each addressed SNP revealed a highly discriminating pattern. Homozygous samples produced clear-cut single base peaks in the expected position, whereas heterozygous counterparts were characterized by distinct half-height peaks representing both allelic positions. Whenever any of the allelic bases of an SNP formed a homopolymer with adjacent bases, the nonallelic signal was added to those of the SNP. This feature did not, however, influence SNP readability. Furthermore, the multibase reading capacity of the described system provides extensive flexibility in regard to the positioning of sequencing primers and allows the determination of several closely located SNPs in a single run.

Circadian Clock-Related Polymorphisms in Seasonal Affective Disorder and their Relevance to Diurnal Preference

“…The PCR products were analyzed with real-time pyrophosphate DNA sequencing (Alderborn et al, 2000), according to standard protocols provided by the manufacturer (Pyrosequencing, Sweden). One of the PCR primers in each pair was 5 0 biotinylated and after denaturing the singlestranded DNA was separated using streptavidin-coated magnetic beads (Dynabeads M-280 Streptavidin from Dynal, Norway) and sequenced with the following sequencing primers: Period2 1244 Gly/Glu; CGATCCTGTGATT-CAAGG, Period3 647 Val/Gly; GGTACCTGTCTCTGGGGGT, and for NPAS2 471 Leu/Ser; CTGCTGTGTGAGGTCGCAG.…”

Poly(ADP-ribose) polymerase-1 polymorphisms, expression and activity in selected human tumour cell lines

“…We isolated DNA directly from the cell lines using a Blood Mini Kit (Qiagen, Crawley, West Sussex, UK) according to the manufacturer's instructions and then genotyped 50 ng of genomic DNA by pyrosequencing using the PSQ96 system (Pyrosequencing, Uppsala, Sweden) as described earlier (Alderborn et al, 2000). Briefly, we amplified the genomic fragment containing the SNP site by PCR with a set of sense and antisense primers (5 0 -AGTCTGTCTCATT CACCAT-3 0 and 5 0 -ATCCTTGCTGCTATCATC-3 0 ) where antisense was 5 0 -biotinylated (Europrim, Invitrogen).…”

A novel CpG-associated brain-expressed candidate gene for chromosome 18q-linked bipolar disorder

“…Sequence variations were identified using the computer software program Polyphred 32 followed by visual inspection. Potential SNPs were confirmed in the BP cases by pyrosequencing using an SNP specific primer 33 on a PSQ96 apparatus (Pyrosequencing AP, Uppsala, Sweden) ( Table 1), and analysed in 160 controls. One set (CCG7) was the result of fragmentation in a (CGG) 6 repeat.…”