An improved strategy for HLA-DRB1 subtyping by digestion of PCR-amplified DNA with allele-specific restriction endonucleases.

Abstract

We developed a simple, rapid and inexpensive method of DRB1 alleles genotyping by digestion of amplified DNA with allele-specific restriction fragments endonucleases. We took advantage of this protocol, initially described by Yunis et al. (1991) and called AFLP (Amplification Length Fragment Polymorphism) to standardise amplification procedure. Typing strategy was particularly studied to limit the number of restriction endonucleases. The determination of DRB1 allele was established on lysed fragments size which allows: (1) the absence of nonidentified allele and (2) a nonambiguous determination of each heterozygous allele. Six specific pairs of primers were chosen to amplify three generic groups: HLA DR 124 (DRB1 1, 2 and 4), HLA DR356810 (DRB1 3, 5, 6, 8 and 10) and DR79 (DRB1 7 and 9) with the same PCR protocol. Forty-eight from the 60 DRB1 alleles may be identified without any ambiguity. With our protocol, the three alleles associated with the most important autoimmune diseases (i.e., DRB1*02, *03 and *04) were totally subtyped. Our amplification procedure is reliable and extremely useful in routine-practice for the study of HLA-DRB1 genotyping of large series of samples and for the determination of DRB1 susceptibility factors involved in different autoimmune diseases.