SNP genotyping by multiplexed solid-phase amplification and fluorescent minisequencing.
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 11 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 5' 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.
Shapero
M H
MH
Affymax Inc., Palo Alto, California 94304, USA.
Leuther
K K
KK
Nguyen
A
A
Scott
M
M
Jones
K W
KW
D004247:DNA / Q000032:analysis / Q000235:genetics; D005456:Fluorescent Dyes / Q000378:metabolism*; D005784:Gene Amplification; D015894:Genome, Human; D005838:Genotype; D006801:Humans; D008863:Microspheres; D021141:Nucleic Acid Amplification Techniques; D009692:Nucleic Acid Heteroduplexes / Q000032:analysis* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*
D005456:Fluorescent Dyes; D009692:Nucleic Acid Heteroduplexes; D004247:DNA