ABSTRACT
This chapter discusses the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method based on the digestion of PCR-amplified DNAs with allele-specific enzymes as a reliable, convenient, and practical HLA class II DNA typing technique, rather than being based on hybridization with multiple sequence-specific oligonucleotide probes (PCR-SSO). The modified PCR-RFLP method incorporating informative enzymes, which have a single recognition site in some alleles but none in other alleles in the amplified regions, is simpler and more sensitive because genotypes can be defined simply by checking whether the amplified DNAs are digested. This modified method facilitates reading the generated RFLP band patterns. The chapter presents the current protocol of PCR-RFLP for HLA class II genotyping and discusses the advantages of PCR-RFLP over the PCR-SSO typing methods. PCR-RFLP can be substituted for the serological and cellular typing methods in HLA matching in transplantation, the HLA-disease association study, paternity testing and functional analysis of HLA antigens in the immunological process.
