SNP genotyping by multiplexed solid-phase amplification and fluorescent minisequencing
SNP genotyping by multiplexed solid-phase amplification and fluorescent minisequencing
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DOI：
10.1101/gr.205001
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发表时间：
2001-11-01
期刊：
影响因子：
7
通讯作者：
Jones, KW
中科院分区：
文献类型：
--
作者：
Shapero, MH;Leuther, KK;Jones, KW
The emerging role of single-nucleotide polymorphisms (SNPs) in clinical association and pharmacogenetic studies has created a need for high-throughput genotyping technologies. We describe a novel method for multiplexed genotyping of SNPs that employs PCR amplification on microspheres. Oligonucleotide PCR primers were designed for each polymorphic locus such that one of the primers contained a recognition site for BbvI (a type IIS restriction enzyme), followed by II nucleotides of locus-specific sequence, which reside immediately upstream of the polymorphic site. Following amplification, this configuration allows for any SNP to be exposed by BbvI digestion and interrogated via primer extension, four-color minisequencing. Primers containing S' acrylamide groups were attached covalently to the solid support through copolymerization into acrylamide beads. Highly multiplexed solid-phase amplification using human genomic DNA was demonstrated with 57 beads in a single reaction. Multiplexed amplification and minisequencing reactions using bead sets representing eight polymorphic loci were carried out with genomic DNA from eight individuals. Sixty-three of 64 genotypes were accurately determined by this method when compared to genotypes determined by restriction-enzyme digestion of PCR products. This method provides an accurate, robust approach toward multiplexed genotyping that may facilitate the use of SNPs in such diverse applications as pharmacogenetics and genome-wide association studies for complex genetic diseases.
