SNP genotyping by multiplexed solid-phase amplification and fluorescent minisequencing

The emerging role of single-nucleotide polymorphisms (SNPs) in clinical ass ociation and pharmacogenetic studies has created a need for high-throughput genotyping technologies. We describe a novel method for multiplexed genoty ping of SNPs that employs PCR amplification on microspheres. Oligonucleotid e PCR primers were designed for each polymorphic locus such that one of the primers contained a recognition site for BbvI (a type IIS restriction enzy me), followed by II nucleotides of locus-specific sequence, which reside im mediately upstream of the polymorphic site. Following amplification, this c onfiguration allows for any SNP to be exposed by BbvI digestion and interro gated via primer extension, four-color minisequencing. Primers containing S ' acrylamide groups were attached covalently to the solid support through c opolymerization into acrylamide beads. Highly multiplexed solid-phase ampli fication using human genomic DNA was demonstrated with 57 beads in a single reaction. Multiplexed amplification and minisequencing reactions using bea d sets representing eight polymorphic loci were carried out with genomic DN A from eight individuals. Sixty-three of 64 genotypes were accurately deter mined by this method when compared to genotypes determined by restriction-e nzyme digestion of PCR products. This method provides an accurate, robust a pproach toward multiplexed genotyping that may facilitate the use of SNPs i n such diverse applications as pharmacogenetics and genome-wide association studies for complex genetic diseases.