通过聚合酶链反应-限制性片段长度多态性（PCR-RFLP）对HLA-DPB1等位基因进行全面分型。

Comprehensive typing of HLA-DPB1 alleles by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).

作者信息

Sengar D P, Hampton N, Goldstein R, Sengar A S

机构信息

Department of Pathology and Laboratory Medicine, Ottawa General Hospital, Ontario.

出版信息

Clin Invest Med. 1995 Dec;18(6):465-72.

PMID:8714790

Abstract

Comprehensive typing of 53 HLA-DPB1 alleles was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method using 78 polymerase chain reaction-sequence-specific oligonucleotide probe (PCR-SSOP) defined DNA specimens (14 retrospective, 64 prospective). A single primer pair was used to amplify the second exon to obtain DPB1-specific amplified product of 294 bp. A combination of RFLPs and cleaved/uncleaved patterns of various endonucleases was employed to resolve DPB1 alleles. A panel of 13 endonucleases (RsaI, Sau96I, BsrBI, DdeI, BsaJI, BssHII, ScaI, ScaI, BbvI, BsgI, FokI, Bsp1286I and BstUI) yielded unique RFLP patterns for all but 2 pairs of DPB1 alleles. However, these remaining 2 pairs of rare alleles could be resolved by an additional digestion with AciI (DPB13901 from 4001 and DPB14901 from 5301). The unique RFLP patterns of 21 DPB1 alleles using PCR-SSOP typed DNA specimens had been verified. Of the 1,378 possible heterozygotic patterns, 69 pairs and a triplet had been identified that would yield identical RFLP patterns. However, all but one pair, DPB1*3901/5301 from 4001/4901, of these heterozygotes could be resolved by double digestions with appropriately selected endonucleases from the panel used here. Thus, PCR-RFLP remains a simple and effective method for high resolution DPB1 typing.

摘要

采用聚合酶链反应-限制性片段长度多态性（PCR-RFLP）方法，使用78个聚合酶链反应-序列特异性寡核苷酸探针（PCR-SSOP）定义的DNA标本（14例回顾性标本，64例前瞻性标本）对53个HLA-DPB1等位基因进行全面分型。使用一对引物扩增第二外显子，以获得294 bp的DPB1特异性扩增产物。采用多种限制性内切酶的限制性片段长度多态性（RFLP）和切割/未切割模式组合来区分DPB1等位基因。一组13种限制性内切酶（RsaI、Sau96I、BsrBI、DdeI、BsaJI、BssHII、ScaI、ScaI、BbvI、BsgI、FokI、Bsp1286I和BstUI）除了2对等位基因外，对所有DPB1等位基因都产生了独特的RFLP模式。然而，这2对剩余的罕见等位基因可以通过用AciI进一步消化来区分（DPB13901与4001区分，DPB14901与5301区分）。使用PCR-SSOP分型的DNA标本对21个DPB1等位基因的独特RFLP模式进行了验证。在1378种可能的杂合模式中，已鉴定出69对和一个三联体，它们会产生相同的RFLP模式。然而，除了一对DPB1*3901/5301（来自4001/4901）外，这些杂合子中的所有其他杂合子都可以通过使用此处所用的一组适当选择的限制性内切酶进行双酶切来区分。因此，PCR-RFLP仍然是一种用于高分辨率DPB1分型的简单有效的方法。