Multiplex genotype determination at a large number of gene loci
Multiplex genotype determination at a large number of gene loci
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DOI：
10.1073/pnas.93.6.2582
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发表时间：
1996-03-19
影响因子：
11.1
通讯作者：
Li, HH
中科院分区：
文献类型：
--
作者：
Lin, ZW;Cui, XF;Li, HH
为了促进大规模基因型分析，开发了一种基于 PCR 的高效多重方法。为了同时扩增大量遗传位点处的靶序列，使用含有 5' 通用尾部的位点特异性引物。在最初的 PCR 步骤中将通用尾部连接到靶序列上，可以用一对与通用尾部相同的引物替换所有特异性引物，并将多重扩增转化为“单重”。描述了使用这种方法同时扩增 26 个基因位点。多重扩增可以与基因型测定结合起来。通过将引物和模板之间的单碱基错配掺入靶序列中，必要时可以将多态性位点转化为所需的限制性片段长度多态性。这样，多态性位点的等位基因PCR产物可以在限制性酶消化后通过凝胶电泳来区分。在本研究中，32个基因座以这种多重方式进行分型。
To facilitate large-scale genotype analysis, an efficient PCR-based multiplex approach has been developed. For simultaneously amplifying the target sequences at a large number of genetic loci, locus-specific primers containing 5' universal tails are used. Attaching the universal tails to the target sequences in the initial PCR steps allows replacement of all specific primers with a pair of primers identical to the universal tails and converts the multiplex amplification into ''uniplex.'' Simultaneous amplification of 26 genetic loci with this approach is described. The multiplex amplification can be coupled with genotype determination. By incorporating a single-base mismatch between a primer and the template into the target sequences, a polymorphic site can be converted into a desirable restriction fragment length polymorphism when it is necessary. In this way, the allelic PCR products for the polymorphic loci can be discriminated by gel electrophoresis after restriction enzyme digestion. In this study, 32 loci were typed in such a multiplex way.