A simple and rapid method for HLA-DQA1 genotyping by digestion of PCR-amplified DNA with allele specific restriction endonucleases.
A simple and rapid method for HLA-DQA1 genotyping by digestion of PCR-amplified DNA with allele specific restriction endonucleases.
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一种简单快速的 HLA-DQA1 基因分型方法，通过等位基因特异性限制性内切酶消化 PCR 扩增的 DNA。
作者：
M. Maeda;N. Murayama;H. Ishii;N. Uryu;M. Ota;K. Tsuji;H. Inoko
应用聚合酶链反应（PCR）技术，从72个HLA纯合B细胞系的基因组DNA中选择性扩增HLA-DQA 1基因的第二外显子。扩增的DNA用Hae Ⅲ、Ddel、ScrFI、Fok Ⅰ和Rsa Ⅰ消化，它们识别DQA 1第二外显子多态性片段中的等位基因序列变异，然后在聚丙烯酰胺凝胶中进行电泳。获得了8种不同的限制性片段多态性模式，其中7种与已知DNA序列预测的模式相同，与血清学分型定义的每个HLA-DQw类型相关。剩下的一种模式无法从序列数据中解释，表明在核苷酸水平上存在新的DQA 1等位基因。该方法简便、快速，可在核苷酸水平上对HLA-DQ分型，无需使用同位素及等位基因特异性寡核苷酸探针，可推广应用于HLA-DR、-Dw DP分型。
The second exon of the HLA-DQA1 genes was selectively amplified from genomic DNAs of 72 HLA-homozygous B cell lines by the polymerase chain reaction (PCR). Amplified DNAs were digested with HaeIII, Ddel, ScrFI, FokI and RsaI, which recognize allelic sequence variations in the polymorphic segments of the DQA1 second exon, and then subjected to electrophoresis in polyacrylamide gels. Eight different polymorphic patterns of restriction fragments were obtained, and seven were identical to patterns predicted from the known DNA sequences, correlating with each HLA-DQw type defined by serological typing. The remaining one pattern cannot be explained from the sequence data, suggesting the presence of a novel DQA1 allele at the nucleotide level. This PCR-RFLP method provides a simple and rapid technique for accurate definition of the HLA-DQ types at the nucleotide level, eliminating the need for radioisotope as well as allele specific oligonucleotide probes and can be extended and applied to HLA-DR, -Dw DP typing.