Multiplex automated primer extension analysis: Simultaneous genotyping of several polymorphisms
Multiplex automated primer extension analysis: Simultaneous genotyping of several polymorphisms
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DOI：
10.2144/01316md05
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发表时间：
2001-12-01
期刊：
影响因子：
2.7
通讯作者：
Reichardt, JKV
中科院分区：
文献类型：
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作者：
Makridakis, NM;Reichardt, JKV
准确、快速的单核苷酸多态性（snp）基因分型对连锁和关联研究具有重要的科学意义。我们在这里报告了一种自动荧光方法，我们称之为多重自动引物扩展分析（MAPA），可以同时准确地对多个已知snp进行基因分型。这是通过大幅改进商用协议（SNaPshot (TM)）来实现的。该方案依赖于引物的延伸，将给定SNP的一个核苷酸5'与荧光双脱氧ntp（迷你测序）连接，然后在ABI PRISM (R) 377半自动DNA测序仪上进行分析。我们的修饰工作原理是将产生引物延伸DNA模板的初始反应和/或在同一引物延伸反应中复用多个引物（对应多个snp）。然后，我们在单个凝胶通道上运行每个多路反应。我们证明，即使在复合杂合子样本中，MAPA也可以同时用于多达四个snp的基因分型，并且具有完全的准确性（基于与测序结果的一致性）。我们还发现，与DNA模板纯化方法不同，引物设计可以显著影响基因分型的准确性，并为快速优化提供了有用的指导方针。
Accurate and fast genotyping of single nucleotide polymorphisms (SNPs) is of significant scientific importance for linkage and association studies. We report here an automated fluorescent method we call multiplex automated primer extension analysis (MAPA) that can accurately genotype multiple known SNPs simultaneously. This is achieved by substantially improving a commercially available protocol (SNaPshot (TM)). This protocol relies on the extension of a primer that ends one nucleotide 5' of a given SNP with fluorescent dideoxy-NTPs (minise-quencing), followed by analysis on an ABI PRISM (R) 377 Semi-Automated DNA Sequencer. Our modification works by multiplexing the initial reaction that produces the DNA template for primer extension and/or multiplexing several primers (corresponding to several SNPs) in the same primer extension reaction. Then, we run each multiplexed reaction on a single gel lane. We demonstrate that MAPA can be used to genotype up to four SNPs simultaneously, even in compound heterozygote samples, with complete accuracy (based on concordance with sequencing results). We also show that primer design, unlike the DNA template purification method, can significantly affect genotyping accuracy, and we suggest useful guidelines for quick optimization.