Deshpande Alina, Valdez Yolanda, Nolan John P
Los Alamos National Laboratory, Los Alamos, New Mexico, USA.
Curr Protoc Cytom. 2005 Nov;Chapter 13:Unit13.4. doi: 10.1002/0471142956.cy1304s34.
Single-nucleotide polymorphisms (SNPs), genome sites with single-base sequence differences between individual chromosomes, are the most common type of genetic variation. Single-base changes can result in disease phenotypes, confer susceptibility or resistance to toxins or pathogens, or serve as markers for distinct allelic segments of DNA, making SNPs an important emerging tool in both basic and clinical research. This unit presents detailed protocols for multiplexed SNP genotyping using primer single-base extension (SBE) adapted to microspheres and flow cytometry. The methods described are best suited for typing a modest number of SNPs in a large number of samples. The Basic Protocol describes extension of the genotyping primers by one nucleotide, a labeled dideoxyribonucleotide that reveals the nucleotide base at that position on the template strand. The extended primers are then captured onto microspheres bearing an oligonucleotide "address" that is the reverse complement of a sequence on the 5' end of the genotyping primers and subsequently measured using flow cytometry.
单核苷酸多态性（SNP）是个体染色体之间具有单碱基序列差异的基因组位点，是最常见的遗传变异类型。单碱基变化可导致疾病表型，赋予对毒素或病原体的易感性或抗性，或作为DNA不同等位基因片段的标记，使SNP成为基础研究和临床研究中一种重要的新兴工具。本单元介绍了使用适用于微球和流式细胞术的引物单碱基延伸（SBE）进行多重SNP基因分型的详细方案。所描述的方法最适合于对大量样本中的少量SNP进行分型。基本方案描述了基因分型引物通过一个核苷酸的延伸，该核苷酸是一个标记的双脱氧核糖核苷酸，揭示模板链上该位置的核苷酸碱基。然后将延伸的引物捕获到带有寡核苷酸“地址”的微球上，该“地址”是基因分型引物5'端序列的反向互补序列，随后使用流式细胞术进行测量。
