Direct analysis of single‐nucleotide polymorphism on double‐stranded DNA by pyrosequencing

Abstract

Pyrosequencing, a new method for DNA sequencing, is gaining widespread use for many different types of DNA analysis. The method takes advantage of four coupled enzymes in a single tube assay to monitor DNA synthesis in real time using a luminometric detection system. Here, we demonstrate the use of pyrosequencing for direct analysis of single-nucleotide polymorphism on double-stranded PCR product. Pyrosequencing data on the human glutathione peroxidase gene (GPX1) from several individuals were analysed and three different allelic variants were determined and confirmed. The possibility of further simplifying the sequencing and template-preparation steps is discussed.

Quantitative profiling of codon 816 KIT mutations can aid in the classification of systemic mast cell disease

“…Pyrosequencing is a DNA sequencing-by-synthesis method developed for single-nucleotide polymorphism analysis 7 that we have adapted here to provide quantitation of the levels of mutated and unmutated product at codon 816 of KIT. For this study, genomic DNA was isolated from BM core biopsy and/or BM aspirate samples from 39 patients with morphological evidence of MCD using WHO criteria, 6 and subjected to pyrosequencing of KIT exon 17.…”

Direct amplification of single-stranded DNA for pyrosequencing using linear-after-the-exponential (LATE)–PCR

“…While several previous reports [24][25][26] have described the use of double-stranded DNA templates for pyrosequencing, most pyrosequencing is carried out using single-stranded templates to boost reaction efficiency by preventing complementary strands from competing with the sequencing primer for hybridization to the template strand. LATE-PCR generates more single-stranded product than double-strand product, thus making it an attractive method for generating pyrosequencing substrate without an additional strand-separation step.…”

“…One group has reported successful sequencing of double-stranded PCR products after removing interfering PCR reaction components with enzymes and/or blocking oligonucleotides [24][25][26]. Although attractive in principle, this method has not come into widespread use, presumably because of the difficulty optimizing each reaction to yield adequate and consistent signals [27].…”

A novel method for KIR-ligand typing by pyrosequencing to predict NK cell alloreactivity

“…It is emerging as an innovative method that may out perform other common methods for SNP analysis such as the use of hybridization probes, endonuclease restriction digestion, discrimination of mismatched DNA ligases or polymerases, allele-specific enzymatic cleavage and DNA sequencing [22]. It is a method where nucleotides are incorporated sequentially from a sequencing primer, differing from other sequencing methods in the order of nucleotide incorporation.…”