Single-nucleotide polymorphism analysis by pyrosequencing

There is a growing demand for high-throughput methods for analysis of singl e-nucleotide polymorphic (SNP) positions. Here, we have evaluated a novel s equencing approach, pyrosequencing, for such purposes. Pyrosequencing is a sequencing-by-synthesis method in which a cascade of enzymatic reactions yi elds detectable light, which is proportional to incorporated nucleotides. O ne feature of typing SNPs with pyrosequencing is that each allelic variant will give a unique sequence compared to the two other variants. These varia nts can easily be distinguished by a pattern recognition software. The soft ware displays the allelic: alternatives and allows for direct comparison wi th the pyrosequencing raw data. For optimal determination of SNPs, various protocols of nucleotide dispensing order were investigated. Here, we demons trate that typing of SNPs can efficiently be performed by pyrosequencing us ing an automated system for parallel analysis of 96 samples in approximatel y 5 min, suitable for large-scale screening and typing of SNPs. (C) 2000 Ac ademic Press.