Techniques used to define human MHC antigens: restriction fragment length polymorphisms.
Polymorphisms within the HLA-DRB1, -DRB3, -DQB1 and -DQ A1 genes are detectable using restriction fragment length polymorphism (RFLP) analysis. DNA is isolated from EDTA-treated blood or from spleen or lymph nodes. The DNA is digested to completion with the restriction endonuclease TaqI and resolved using agarose gel electrophoresis. The DNA after denaturation is then transferred to a nylon membrane (Southern blotting) and hybridised with radiolabelled cDNA probes: HLA-DR beta pRTV1, HLA-DQ beta pII-beta-1 and HLA-DQ alpha pDCH1. After autoradiography the membrane is dehybridised prior to rehybridisation. This system is very useful in those situations where serological assignment is difficult due to poor quality or low numbers of circulating B cells and where there is a lack of reliable antisera for certain specificities. The RFLP techniques can also define subtypes of DR and DQ serological specificities. However, certain alleles have the same RFLP. In some instances by identifying the DQ allele the DR allele can be determined by association due to linkage disequilibrium (e.g., DRw17-Dw25-DQw2 and DRw13-Dw25-DQw6). In other instances (e.g., DR1 and DRBr), the problem can be resolved using serology. In addition the RFLP system cannot be applied prospectively to the cadaver donor situation because of time restrictions. Thus the RFLP system complements existing serological techniques. However, it can be very useful as a quality control for the serological methods especially in the assessment of the quality of antisera and in the determination of discrepancies between centres.(ABSTRACT TRUNCATED AT 250 WORDS)
Middleton
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Northern Ireland Tissue Typing Laboratory, Belfast City Hospital, U.K.
D000483:Alleles; D004247:DNA / Q000032:analysis / Q000302:isolation & purification; D015342:DNA Probes; D005838:Genotype; D006680:HLA Antigens / Q000032:analysis* / Q000235:genetics; D006683:HLA-DQ Antigens / Q000032:analysis; D006684:HLA-DR Antigens / Q000032:analysis; D006801:Humans; D016030:Kidney Transplantation / Q000276:immunology; D012150:Polymorphism, Restriction Fragment Length
D015342:DNA Probes; D006680:HLA Antigens; D006683:HLA-DQ Antigens; D006684:HLA-DR Antigens; D004247:DNA