Ota M, Seki T, Fukushima H, Tsuji K, Inoko H
Department of Legal Medicine, Shinshu University School of Medicine, Nagano, Japan.
Tissue Antigens. 1992 Apr;39(4):187-202. doi: 10.1111/j.1399-0039.1992.tb01935.x.
We previously introduced HLA-DQA1, -DPB1 and DQB1 genotyping with the modified PCR-RFLP method using some informative restriction enzymes which have either a single cleavage site or alternatively no cleavage site in the amplified DNA region, depending on the HLA alleles, making reading of RFLP band patterns much easier. In this study, 43 HLA-DRB1 alleles, excluding DRB11103 and 1104 for which no restriction enzymes are available to distinguish each from the other, could be defined by this modified PCR-RFLP method combined with 7 pairs of group-specific primers. It is impossible to distinguish DRB10701 and DRB10702 as they are identical for the second exon of DRB1. For DR1-DRB1, DR2-DRB1, DR4-DRB1, DR7-DR1, DR9-DRB1, DRw10-DRB1 or DRw52 associated antigens (DR3, w11, w12, w13, w14, and DRw8)-DRB1 gene amplification, the second exon of the DRB1 gene was selectively amplified using each group-specific primer from genomic DNAs of 70 HLA-homozygous B-cell lines and healthy Japanese by PCR. Amplified DNAs were digested with restriction endonucleases and then subjected to electrophoresis assaying simply for cutting, or no cutting, of the DNA, although some alleles can be distinguished only after examination of RFLP band patterns generated and in some cases using double digestion technique with two restriction enzymes. This modified PCR-RFLP method can be successfully applied to all possible DRB1 heterozygotes, despite the fact that 15 pairs of heterozygotes among them cannot be distinguished theoretically by the PCR-SSO method, because the PCR-RFLP method can tell whether two polymorphic sites are linked to each other (cis position) or located on a different chromosome (trans position) by checking the length of RFLP bands generated with double digestion. Thus, the PCR-RFLP method is technically simple, practical and inexpensive for determination of the HLA-DRB1 alleles for routine HLA typing work.
我们之前介绍了使用改良的PCR-RFLP方法进行HLA-DQA1、-DPB1和DQB1基因分型，该方法使用了一些信息丰富的限制性内切酶，这些酶在扩增的DNA区域根据HLA等位基因的不同，要么有单个切割位点，要么没有切割位点，这使得RFLP条带模式的读取更加容易。在本研究中，通过这种改良的PCR-RFLP方法结合7对组特异性引物，可以定义43个HLA-DRB1等位基因，不包括没有限制性内切酶可用于区分彼此的DRB11103和1104。由于DRB1的第二个外显子对于DRB10701和DRB10702是相同的，所以无法区分它们。对于DR1-DRB1、DR2-DRB1、DR4-DRB1、DR7-DR1、DR9-DRB1、DRw10-DRB1或DRw52相关抗原（DR3、w11、w12、w13、w14和DRw8）-DRB1基因扩增，使用每组特异性引物从70个HLA纯合B细胞系的基因组DNA和健康日本人的基因组DNA中通过PCR选择性扩增DRB1基因的第二个外显子。扩增的DNA用限制性内切酶消化，然后进行电泳，仅检测DNA的切割或未切割情况，尽管有些等位基因只有在检查产生的RFLP条带模式后，以及在某些情况下使用两种限制性内切酶的双重消化技术才能区分。这种改良的PCR-RFLP方法可以成功应用于所有可能的DRB1杂合子，尽管其中15对杂合子理论上不能通过PCR-SSO方法区分，因为PCR-RFLP方法可以通过检查双重消化产生的RFLP条带的长度来判断两个多态性位点是彼此连锁（顺式位置）还是位于不同的染色体上（反式位置）。因此，PCR-RFLP方法在技术上简单、实用且成本低廉，可用于常规HLA分型工作中HLA-DRB1等位基因的测定。
