High-throughput genotyping of single nucleotide polymorphisms with high sensitivity.
High-throughput genotyping of single nucleotide polymorphisms with high sensitivity.
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高灵敏度单核苷酸多态性的高通量基因分型。
DOI：
10.1007/978-1-59745-515-2_18
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发表时间：
2007
期刊：
影响因子：
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通讯作者：
Gao,Richeng
中科院分区：
文献类型：
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作者：
Li,Honghua;Wang,Hui-Yun;Cui,Xiangfeng;Luo,Minjie;Hu,Guohong;Greenawalt,DanielleM;Tereshchenko,IrinaV;Li,JamesY;Chu,Yi;Gao,Richeng
分析由单核苷酸多态性（SNPs）组成的大量遗传标记的能力可能会使人类生物学的认识取得重大进展。最近几种高通量基因分型方法的发展极大地促进了大规模SNP分析。然而，由于它们的灵敏度相对较低，这些方法的应用，特别是在涉及少量材料的研究中，一直受到限制。本章详细介绍了高通量、高灵敏度基因分型系统的实验步骤。该系统涉及使用计算机程序选择引物，预计在PCR扩增期间不会产生大量的非特异性产物。PCR后，一小部分PCR产物作为模板生成单链DNA （ssDNA）。来自不同SNP位点的ssDNA序列然后通过将这些序列杂交到排列在玻璃表面的探针上来分辨。探针被设计成这样一种方式，即与ssDNA模板杂交时，将它们的3'端放置在多态性位点旁边。因此，探针可以用荧光标记的染料终止物以等位基因特异性的方式进行标记。snp的等位基因状态可以通过分析与相应探针结合的不同荧光颜色的数量来确定。该基因分型系统准确度高，能够分析单个单倍体细胞中的1000个snp。
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.