Hirschhorn J N, Sklar P, Lindblad-Toh K, Lim Y M, Ruiz-Gutierrez M, Bolk S, Langhorst B, Schaffner S, Winchester E, Lander E S
Whitehead Institute/MIT Center for Genome Research, One Kendall Square, Building 300, Cambridge, MA 02139, USA.
Proc Natl Acad Sci U S A. 2000 Oct 24;97(22):12164-9. doi: 10.1073/pnas.210394597.
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 1,000,000 within a year. Rather, progress is limited by the inability to genotype large numbers of SNPs. Current genotyping methods are suitable for studying individual loci or at most a handful at a time. Here, we describe a method for parallel genotyping of SNPs, called single base extension-tag array on glass slides, SBE-TAGS. The principle is as follows. SNPs are genotyped by single base extension (SBE), using bifunctional primers carrying a unique sequence tag in addition to a locus-specific sequence. Because each locus has a distinct tag, the genotyping reactions can be performed in a highly 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. SBE-TAGS is simple and inexpensive because of the high degree 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.
生成人类单核苷酸多态性（SNP）已不再是疾病基因研究的限速步骤。公共数据库中的SNP数量已超过200,000个，预计一年内总数将超过1,000,000个。相反，研究进展受到无法对大量SNP进行基因分型的限制。当前的基因分型方法适用于研究单个位点，或者一次最多研究少数几个位点。在此，我们描述了一种用于SNP平行基因分型的方法，称为玻片上的单碱基延伸标签阵列，即SBE-TAGS。其原理如下。通过单碱基延伸（SBE）对SNP进行基因分型，使用除了位点特异性序列外还携带独特序列标签的双功能引物。由于每个位点都有一个独特的标签，基因分型反应可以以高度多重化的方式进行，然后通过与排列在玻片上的序列标签的反向互补序列杂交，对所得产物进行“解复用”。由于高度多重化以及使用易于生成的通用标签阵列，SBE-TAGS简单且成本低廉。该方法也具有高度准确性：我们对100多个SNP进行了基因分型，获得了5000多个基因型，准确率约为99%。
