SBE-TAGS: An array-based method for efficient single-nucleotide polymorphism genotyping

Generating human single-nucleotide polymorphisms (SNPs) is no longer a rate -limiting step for genetic studies of disease. The number of SNPs in public databases already exceeds 200,000, and the total is expected to exceed 1,0 00.000 within a year. Rather, progress is limited by the inability to genot ype large numbers of SNPs. Current genotyping methods are suitable for stud ying individual loci or at most a handful at a time. Here, we describe a me thod for parallel genotyping of SNPs, called single base extension-tag arra y on glass slides, SEE-TAGS. The principle is as follows. SNPs are genotype d by single base extension (SBE), using bifunctional primers carrying a uni que sequence tag in addition to a locus-specific sequence. Because each loc us has a distinct tag, the genotyping reactions can be performed in a highl y multiplexed fashion, and the resulting product can then be "demultiplexed " by hybridization to the reverse complements of the sequence tags arrayed on a glass slide. SEE-TAGS is simple and inexpensive because of the high de gree of multiplexing and the use of an easily generated, generic tag array. The method is also highly accurate: we genotyped over 100 SNPs, obtaining over 5,000 genotypes, with approximately 99% accuracy.