Gyllensten U B, Erlich H A
Department of Human Genetics, Cetus Corporation, Emeryville, CA 94608.
Proc Natl Acad Sci U S A. 1988 Oct;85(20):7652-6. doi: 10.1073/pnas.85.20.7652.
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.
单拷贝序列可通过聚合酶链反应从基因组DNA中进行酶促扩增。通过使用不等摩尔量的两种扩增引物，有可能在一步反应中扩增一个单拷贝基因，并产生过量的选定链的单链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来直接测序。通过使用这些方法，我们研究了HLA-DQA基因座的等位基因多样性及其与血清学定义的HLA-DR和-DQ类型的关联。该分析共揭示了八个等位基因和另外三种单倍型。该方法在筛选人类基因突变方面有广泛应用，并有助于将基因的酶促扩增与自动测序相联系。
