GENERATION OF SINGLE-STRANDED-DNA BY THE POLYMERASE CHAIN-REACTION AND ITS APPLICATION TO DIRECT SEQUENCING OF THE HLA-DQA LOCUS
GENERATION OF SINGLE-STRANDED-DNA BY THE POLYMERASE CHAIN-REACTION AND ITS APPLICATION TO DIRECT SEQUENCING OF THE HLA-DQA LOCUS
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DOI：
10.1073/pnas.85.20.7652
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发表时间：
1988-10-01
影响因子：
11.1
通讯作者：
ERLICH, HA
中科院分区：
文献类型：
--
作者：
GYLLENSTEN, UB;ERLICH, HA
单拷贝序列可以通过聚合酶链反应从基因组DNA中酶扩增出来。通过使用不等摩尔量的两种扩增引物，可以在一个步骤中扩增单拷贝基因并产生多余的单链DNA，用于直接测序或用作杂交探针。此外，杂合子中的单个等位基因可以通过在扩增反应中使用等位基因特异性寡核苷酸或作为测序引物直接测序。通过这些方法，我们研究了HLA-DQA位点的等位基因多样性及其与血清学定义的HLA-DR和-DQ型的关系。该分析共揭示了8个等位基因和3个额外的单倍型。该方法在筛选人类基因突变方面具有广泛的应用，并促进了基因酶扩增与自动测序的联系。
Single-copy sequences can be enzymatically amplified from genomic DNA by the polymerase chain reaction. By using unequal molar amounts of the two amplification primers, it is possible in a single step to amplify a single-copy gene and produce an excess of single-stranded DNA of a chosen strand for direct sequencing or for use as a hybridization probe. Further, individual alleles in a heterozygote can be sequenced directly by using allele-specific oligonucleotides either in the amplification reaction or as sequencing primers. By using these methods, we have studied the allelic diversity at the HLA-DQA locus and its association with the serologically defined HLA-DR and -DQ types. This analysis has revealed a total of eight alleles and three additional haplotypes. This procedure has wide applications in screening for mutations in human genes and facilitates the linking of enzymatic amplification of genes to automated sequencing.