1. An overview of the restriction fragment length polymorphism of the HLA-D region: its application to individual D-, DR- typing by computerized analyses
- Author
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Mara v.d. Bijl, Marja C. J. A. Eggermond, J. D'Amaro, Fei Hongming, Marius J. Giphart, Geziena M.Th. Schreuder, René R. P. Vries, and Marcel G. J. Tilanus
- Subjects
Paper ,Immunology ,Biochemistry ,Restriction fragment ,Complementary DNA ,Methods ,Genetics ,medicine ,Humans ,Immunology and Allergy ,Typing ,Southern blot ,HLA-D Antigens ,Polymorphism, Genetic ,biology ,medicine.diagnostic_test ,Computers ,Histocompatibility Testing ,Collodion ,DNA ,HLA-DR Antigens ,General Medicine ,Molecular biology ,Blot ,Restriction enzyme ,biology.protein ,Electrophoresis, Polyacrylamide Gel ,Restriction fragment length polymorphism ,Tissue typing ,Polymorphism, Restriction Fragment Length ,Software - Abstract
HLA-D/DR alleles as defined by cellular and serological typing can also be identified by biochemical methods. The Southern blot technique provides an additional typing facility which can be applied to DNA obtained from any source of nucleated cells. The polymorphism revealed by Southern blot analyses, the so-called restriction fragment length polymorphism (RFLP), depends upon the restriction enzyme and cDNA probes used. To identify HLA-DR specificities a protocol was developed based on the use of the results of southern blot analyses with several restriction enzymes and cDNA probes within a panel of HLA-D/DR homozygous cells representing the DR1 to DRw8 alleles. First, hybridizations with the 3' untranslated sequence of the DR beta cDNA probe, after digestion of the DNA with PvuII (PvuII-DR beta 3') allows the selective identification of DR1, DR2 and DRw8; DR3, DR5 and DRw6 are found as one group as well as DR4 and DR7 as another. Second, TaqI-DQ alpha hybridization allows the splitting of DRw6-Dw18, DRw6-Dw19 and DRw6-Dw9 from the DR3, DR5 and DRw6 group. The other alleles DR3, DR4, DR5, DRw6-Dw16 and DR7 are revealed by dehybridization and rehybridization of the blot with a DR beta cDNA probe. This protocol was used to test whether in a panel of 30 randomly chosen individuals the HLA-DR typing could be performed. The results were highly concordant to the serotyping. Furthermore by adding the Pst-DR beta and TaqI-DQ alpha RFLPs, most of the MLC defined Dw specificities could also be identified. An overview of the specific fragments described here has been summarized in matrices which can be used as references for DNA-typing in computerized analyses.
- Published
- 2008