A nested PCR‐RFLP method for high‐resolution typing of HLA‐A alleles

Abstract

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: A*0201/0207/0215N/0220/0222, A*0205/0208/0214, A*0206/0221, A*0212/0213, A*2402/2405, A*2406/2413, A*2601/2605, A*2603/2606 and A*7401/7402. 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. A*0201 and A*0207 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 A*0201/0220/0222 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.

Preimplantation HLA typing for stem cell transplantation treatment of congenital and acquired bone marrow failures

“…as mentioned, to avoid late complications due to severe GVH [3][4][5]. A total of 23 couples at risk for producing a progeny with FA presented for preimplantation HLA typing, including carriers of IVS 4+4 A-T mutation in FANCC gene, carriers of FANCD2, FANCF, FANCI, FAMCCJ, and FANCA gene mutations [22][23][24][25][26][27]. Overall, 63 cycles were performed, resulting in transfer of 61 unaffected HLA matched embryos in 41 cycles, yielding twelve unaffected pregnancies and birth of nine FA free and HLA matched children, as potential donors for their siblings.…”

Novel classical MHC class I alleles identified in horses by sequencing clones of reverse transcription‐PCR products

“…Molecular typing procedures are required to clearly define individual MHC class I alleles. Current procedures for molecular typing include DNA sequencing, PCR using type‐specific primers, restriction fragment length polymorphism (RFLP), single strand conformation polymorphism (SSCP) and reference strand mediated conformation analysis (RSCA) (Arguello et al ., 1998; Mitsunaga et al ., 1998; Katsuyama et al ., 1999; Kurz et al ., 1999). Even though these methods are more specific than serological methods, RFLP or SSCP analysis of PCR amplicons may not completely differentiate PCR artifacts nor detect some point mutations of classical MHC class I genes.…”

Analysis of 66 patients definitive with transfusion‐associated graft‐versus‐host disease and the effect of universal irradiation of blood

“…Previously, it was very difficult to type HLA before and after the development of TA‐GVHD, because pancytopenia in this disorder progresses so rapidly that there are not sufficient lymphocytes for HLA typing. Therefore, we typed HLA antigens mainly using polymerase chain reaction (PCR) amplification technologies (Mitsunaga et al , , ).…”