PCR-Restriction Fragment Length Polymorphism Typing of Class I and II Alleles

Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) as a method for the definition of human leukocyte antigen (HLA ) alleles has most often been applied as a typing technique for HLA class II . The best and most comprehensive overview is provided by Inoko and Ota (1 ). Methods have also been published for HLA-C (2 ) and a nested PCR-RFLP method for high resolution definition of HLA-A (3 ). The technique consists of the generic amplification of an HLA locus or subregion by PCR amplification, followed by restriction enzyme digestion of the amplified products and gel electrophoresis with ethidium bromide to visualize the fragments. Acrylamide gels are preferred for the separation of small fragments. As the nucleotide sequence of alleles and therefore their restriction enzyme cut sites are known, careful selection of specific restriction enzymes that will cut certain alleles and not others can allow fine definition of HLA alleles.