RAPID CLONING OF HLA-A,B CDNA BY USING THE POLYMERASE CHAIN-REACTION - FREQUENCY AND NATURE OF ERRORS PRODUCED IN AMPLIFICATION
RAPID CLONING OF HLA-A,B CDNA BY USING THE POLYMERASE CHAIN-REACTION - FREQUENCY AND NATURE OF ERRORS PRODUCED IN AMPLIFICATION
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DOI：
10.1073/pnas.87.7.2833
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发表时间：
1990-04-01
影响因子：
11.1
通讯作者：
PARHAM, P
中科院分区：
文献类型：
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作者：
ENNIS, PD;ZEMMOUR, J;PARHAM, P
本文报道了用聚合酶链反应（PCR）克隆HLA-A、B全长cDNA（1.1个酶）的方法。克隆了6个HLA-A、B等位基因（HLA-A2、-A25、-B7、-B37、-B51和-B57），并测定了它们的结构。对每个等位基因的多个PCR克隆进行测序，以获得准确的共有序列和具有该序列的“真实”克隆。来自50个克隆的编码5个不同等位基因的序列允许评估PCR产生的错误的频率和性质。这些包括重组、缺失和插入以及点置换。以30%和70%之间的频率获得真实的克隆，并且三个或四个克隆的分析通常应足以表征等位基因。
A method for cloning full-length HLA-A,B cDNA (1.1 kilobases) by using the polymerase chain reaction (PCR) is described. Six HLA-A,B alleles (HLA-A2, -A25, -B7,-B37, -B51, and -B57) were cloned, and their structures were determined. Multiple PCR clones for each allele were sequenced to obtain both an accurate consensus sequence and an "authentic" clone having that sequence. Sequences from 50 clones encoding five different alleles permit assessment of the frequency and nature of PCR-produced errors. These include recombinations, deletions, and insertions in addition to point substitutions. Authentic clones were obtained at a frequency of between 30% and 70%, and analysis of three or four clones generally should be sufficient for characterization of an allele.