Li Honghua, Wang Hui-Yun, Cui Xiangfeng, Luo Minjie, Hu Guohong, Greenawalt Danielle M, Tereshchenko Irina V, Li James Y, Chu Yi, Gao Richeng
Methods Mol Biol. 2007;396:281-94. doi: 10.1007/978-1-59745-515-2_18.
The ability to analyze a large number of genetic markers consisting of single nucleotide polymorphisms (SNPs) may bring about significant advance in understanding human biology. Recent development of several high-throughput genotyping approaches has significantly facilitated large-scale SNP analysis. However, because of their relatively low sensitivity, application of these approaches, especially in studies involving a small amount of material, has been limited. In this chapter, detailed experimental procedures for a high-throughput and highly sensitive genotyping system are described. The system involves using computer program selected primers that are expected not to generate a significant amount of nonspecific products during PCR amplification. After PCR, a small aliquot of the PCR product is used as templates to generate single-stranded DNA (ssDNA). ssDNA sequences from different SNP loci are then resolved by hybridizing these sequences to the probes arrayed onto glass surface. The probes are designed in such a way that hybridizing to the ssDNA templates places their 3'-ends next to the polymorphic sites. Therefore, the probes can be labeled in an allele-specific way using fluorescently labeled dye terminators. The allelic states of the SNPs can then be determined by analyzing the amounts of different fluorescent colors incorporated to the corresponding probes. The genotyping system is highly accurate and capable of analyzing >1000 SNPs in individual haploid cells.
分析由单核苷酸多态性（SNP）组成的大量遗传标记的能力，可能会在理解人类生物学方面带来重大进展。几种高通量基因分型方法的最新发展极大地促进了大规模SNP分析。然而，由于它们相对较低的灵敏度，这些方法的应用受到了限制，尤其是在涉及少量材料的研究中。在本章中，将描述一种高通量、高灵敏度基因分型系统的详细实验步骤。该系统涉及使用计算机程序选择的引物，这些引物预计在PCR扩增过程中不会产生大量非特异性产物。PCR后，取一小份PCR产物作为模板生成单链DNA（ssDNA）。然后，通过将来自不同SNP位点的ssDNA序列与排列在玻璃表面的探针杂交来解析这些序列。探针的设计方式是，与ssDNA模板杂交会使它们的3'端位于多态性位点旁边。因此，可以使用荧光标记的染料终止剂以等位基因特异性方式标记探针。然后，通过分析掺入相应探针的不同荧光颜色的量来确定SNP的等位基因状态。该基因分型系统高度准确，能够分析单个单倍体细胞中的>1000个SNP。
