High-resolution analysis of the human HLA-DR polymorphism by hybridization with sequence-specific oligonucleotide probes.

Abstract

The human major histocompatibility complex class II antigens of the HLA-D are highly polymorphic, surface proteins essential in the cellular interactions necessary for an immune response. The analysis of this polymorphism is crucial for (i) histocompatibility matching for transplantation and (ii) understanding the association between HLA-D and certain important diseases. The polymorphism of certain HLA-D haplotypes may escape detection by current methodologies. Analysis at the genomic level of the polymorphism of one of the HLA-D subregions HLA-DR, using oligonucleotide probes specific for the polymorphic regions, is capable of distinguishing single nucleotide differences. The DRw6 haplotype was analyzed in view of the lack of DRw6 specific sera. On the basis of nucleotide sequence analysis, the DRw6 haplotype consists of at least two subtypes. When analyzed with oligonucleotide probes, this split identifies new polymorphic groups that differ from the DRw6 serological subgroups.

Polymorphisms of the Immune Response Genes:selected Biological, Methodical and Medical Aspects

“…In PCR-SSO (Polymerase chain reaction with sequence specific oligonucleotides), the DNA amplified by PCR is transferred to suitable surface (dot-blot). Labelled sequence specific probes are hybridised to DNA and then detected only in the case of total complementarity 16 . The probes are visualised by colour reaction or fluorescence.…”

Analysis of HLA-D micropolymorphism by a simple procedure: RNA oligonucleotide hybridization.

“…2, where the RNA from two DR3 homozygous lines (AVL and QBL) is analyzed. We have shown by DNA sequence analysis that these two cells differ at their DR BIII (DRw52) locus, where one finds, respectively, the DRw52a and DRw52b alleles (6,16,20). The 52a and 52b oligonucleotides differ by only one out of 19 nucleotides and there is complete discrimination in the RNA hybridization (Fig.…”

“…Indeed many oligonucleotide probes hybridize, as expected, to one or more other non-HLA and nonpolymorphic bands. These can easily be distinguished on an electrophoretic blot (16) Preparation and blotting of RNA. RNA was prepared from cell pellets (fresh or frozen) using the guanidine isothiocyanate/CsCl gradient procedure (18).…”

“…These structural data have also lead us to propose an alternative approach to HLA genotyping: synthetic oligonucleotides, corresponding to specific variable regions of the class II genes can be used as hybridization probes for a direct analysis ofphenotypically relevant polymorphism at the DNA sequence level (16). This procedure is now being used not only to refine HLA typing beyond the capabilities of other procedures including RFLP, but also to establish correlations between individual DNA segments and the functional or serological specificities of individual class II gene products.…”

“…DNA oligonucleotide typing (DOT), as described by Angelini et al (16), is sensitive, accurate, and informative. However, it requires the preparation of DNA and the electrophoretic fractionation of restriction enzyme-digested DNA before hybridization.…”