Mitsunaga S, Tokunaga K, Kashiwase K, Akaza T, Tadokoro K, Juji T
Department of Research, Japanese Red Cross Central Blood Centre, Tokyo, Japan.
Eur J Immunogenet. 1998 Feb;25(1):15-27. doi: 10.1046/j.1365-2370.1998.00093.x.
We developed a nested polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) method for high-resolution typing of HLA-A alleles. HLA-A alleles can be identified by this method without the need for other information such as serological type. The first PCR was performed using outer primers, ASP5 and ASP3, specific for the HLA-A gene, and a 991-bp DNA fragment extending from exon 1 through exon 3 was amplified. In the second PCRs, exon 2 and exon 3 of the HLA-A gene were amplified separately from the diluted first PCR product using nested primers. Computer analysis of cleavage patterns for 78 HLA-A alleles showed that 31 RFLP patterns could be obtained by digestion of the exon 2 PCR product using eight restriction endonucleases and 42 RFLP patterns by digestion of the exon 3 PCR product using 11 restriction endonucleases, and all alleles could be discriminated based on combinations of these RFLP patterns except for nine allele groups or pairs: A0201/0207/0215N/0220/0222, A0205/0208/0214, A0206/0221, A0212/0213, A2402/2405, A2406/2413, A2601/2605, A2603/2606 and A7401/7402. Thus, 65 PCR-RFLP patterns were predicted from the results of analysis of digestion patterns of 78 HLA-A alleles. Among 2145 possible homozygous and heterozygous combinations of the 65 distinguishable PCR-RFLP patterns, 82 combinations were predicted to have the same PCR-RFLP patterns. In PCR-RFLP analysis, although the nested primers were not specific for the HLA-A gene, clear RFLP banding patterns were obtained because specificity was guaranteed by the use of the outer primers, ASPS and ASP3 in the first PCR. A0201 and A0207 occur relatively frequently in the Asian populations among indistinguishable allele groups or pairs using the present PCR-RFLP method. We also developed a PCR sequence-specific primers (PCR-SSP) method for distinguishing between A0201/0220/0222 and A*0207/0215N. We could identify 39 alleles (groups) upon HLA-A typing of 50 Japanese individuals, 40 cell lines of the Fourth Asia-Oceania Histocompatibility Workshop, and 80 cell lines of the UCLA International Cell Exchange Program using the present PCR-RFLP and PCR-SSP methods.
我们开发了一种巢式聚合酶链反应（PCR）-限制性片段长度多态性（RFLP）方法，用于对HLA - A等位基因进行高分辨率分型。通过这种方法可以识别HLA - A等位基因，而无需血清学类型等其他信息。第一次PCR使用针对HLA - A基因的外部引物ASP5和ASP3进行，扩增出一个从外显子1延伸至外显子3的991bp DNA片段。在第二次PCR中，使用巢式引物从稀释后的第一次PCR产物中分别扩增HLA - A基因的外显子2和外显子3。对78个HLA - A等位基因的酶切图谱进行计算机分析表明，使用8种限制性内切酶消化外显子2的PCR产物可获得31种RFLP图谱，使用11种限制性内切酶消化外显子3的PCR产物可获得42种RFLP图谱，除了9个等位基因组或等位基因对：A0201/0207/0215N/0220/0222、A0205/0208/0214、A0206/0221、A0212/0213、A2402/2405、A2406/2413、A2601/2605、A2603/2606和A7401/7402外，所有等位基因均可根据这些RFLP图谱的组合进行区分。因此，根据78个HLA - A等位基因的酶切图谱分析结果预测出65种PCR - RFLP图谱。在65种可区分的PCR - RFLP图谱的2145种可能的纯合和杂合组合中，预测有82种组合具有相同的PCR - RFLP图谱。在PCR - RFLP分析中，虽然巢式引物并非HLA - A基因特异性的，但由于在第一次PCR中使用了外部引物ASPS和ASP3保证了特异性，所以获得了清晰的RFLP条带图谱。在使用本PCR - RFLP方法无法区分的等位基因组或等位基因对中，A0201和A0207在亚洲人群中出现频率相对较高。我们还开发了一种PCR序列特异性引物（PCR - SSP）方法来区分A0201/0220/0222和A*0207/0215N。使用本PCR - RFLP和PCR - SSP方法对50名日本个体、第四届亚洲 - 大洋洲组织相容性研讨会的40个细胞系以及加州大学洛杉矶分校国际细胞交换项目的80个细胞系进行HLA - A分型时，我们能够识别出39个等位基因（组）。
