HLA‐DRB1 genotyping by modified PCR‐RFLP method combined with group‐specific primers

Abstract

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 DRB1*1103 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 DRB1*0701 and DRB1*0702 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.

High-resolution HLA-DRB1 and DQB1 genotyping in Japanese patients with testicular germ cell carcinoma

“…High-resolution genotyping is essential because the polymorphism of the peptidebinding domain of MHC class II molecules is more precisely determined by genotypes than by serotypes. The HLA-DRBI and -DQBI genes are extremely polymorphic, and the amino acids they encode are located in regions that line the sides and floor of the peptidebinding groove, which is related to immune responsiveness as well as to the ability to bind antigenic peptide and to be recognized by CD4 T-cell receptors (Rukstalis et al, 1989;Nomura et al, 1991;Ota et al, 1992;Ayala, 1995;Futami et al, 1995).…”

“…The modified PCR-RFLP method for HLA-DRB1 genotyping described by Ota et al (1992) and that for HLA-DQB1 described by Nomura et al (1991) were used. Briefly, the polymorphic exon 2 (-) controls.…”

Combination of HLA-A24, -DQA1*03, and -DR9 Contributes to Acute-Onset and Early Complete β-Cell Destruction in Type 1 Diabetes

“…HLA typing. HLA-DR and -DQ alleles were typed by the previously described PCR-restriction fragment-length polymorphism (RFLP) methods (22)(23)(24). HLA-A, -C, -B, and -DR antigens were typed by the microcytotoxicity test (25), except in 38 patients for whom only HLA-A alleles were determined by PCR-RFLP method (26).…”

A methodological appraisal of the impact of different classification procedures used in three different phases of the australian rheumatoid arthritis twin survey

“…Hand radiographs have been obtained on 22 individuals diagnosed as having RA and 3 unaffected co-twins (2 osteoarthritis, 1 arthralgia). HLA-DRB1 alleles were determined by a combination of methods including sequence specific oligonucleotide typing as described in the 1 lth International Histocompatability Workshop (Kimura and Sasazuki [14]): sequencing specific priming (Olerup and Zetterquist [15]): and PCR-RFLP using a modified method from Ota et al [16]. In cases were the zygosity of the twin was questionable, DNA typing with simple tandem repeat (STR) polymorphism markers was performed.…”