Parallel genotyping of human SNPs using generic high-density oligonucleotide tag arrays

Large scale human genetic studies require technologies for generating milli ons of genotypes with relative ease but also at a reasonable cost and with high accuracy. We describe a highly parallel method for genotyping single n ucleotide polymorphisms (SNPs), using generic high-density oligonucleotide arrays that contain thousands of preselected 20-mer oligonucleotide tags. F irst, marker-specific primers are used in PCR amplifications of genomic reg ions containing SNPs. Second, the amplification products are used as templa tes in single base extension (SBE) reactions using chimeric primers with 3' complementarity to the specific SNP loci and 5' complementarity to specifi c probes, or tags, synthesized on the array. The SEE primers, terminating o ne base before the polymorphic site, are extended in the presence of labele d dideoxy NTPs, using a different label for each of the two SNP alleles, an d hybridized to the tag array. Third, genotypes are deduced from the Fluore scence intensity ratio of the two colors. This approach takes advantage of multiplexed sample preparation, hybridization, and analysis at each stage. We illustrate and test this method by genotyping 44 individuals For 142 hum an SNPs identified previously in 62 candidate hypertension genes. Because t he hybridization results are quantitative, this method can also be used for allele-frequency estimation in pooled DNA samples.