Single-nucleotide polymorphism analysis by pyrosequencing

Abstract

There is a growing demand for high-throughput methods for analysis of single-nucleotide polymorphic (SNP) positions. Here, we have evaluated a novel sequencing approach, pyrosequencing, for such purposes. Pyrosequencing is a sequencing-by-synthesis method in which a cascade of enzymatic reactions yields detectable light, which is proportional to incorporated nucleotides. One feature of typing SNPs with pyrosequencing is that each allelic variant will give a unique sequence compared to the two other variants. These variants can easily be distinguished by a pattern recognition software. The software displays the allelic alternatives and allows for direct comparison with the pyrosequencing raw data. For optimal determination of SNPs, various protocols of nucleotide dispensing order were investigated. Here, we demonstrate that typing of SNPs can efficiently be performed by pyrosequencing using an automated system for parallel analysis of 96 samples in approximately 5 min, suitable for large-scale screening and typing of SNPs.