DNA Extraction and PCR-RFLP. High molecularweight genomicDNA

Third, because DRB1 antigens are the most polymorphic and hyper variable of all the HLA antigens, reagents often give cross-reactions. As a result ofthese drawbacks, serological HLA-DRB1 typing is more demanding technically and gives up to 25% typing errors as compared with genotyping methods, making it less reliable (7, 8). Of the several available genotyping methods, DNA-RFLP analysis requires 2—3 days, a large amount of DNA is needed, and the radioactive labeling of the cDNA probes is problematic. Although the PCR-single-strand conformation polymorphism and PCR-sequence-specific oligonucleo tide methods are less time consuming, the establishment of appropri ate conditions and the interpretation of results are troublesome. More over, the PCR-sequence-specific oligonucleotide method theoretically cannot distinguish 15 pairs of heterozygotes, and the adjustment of the oligonucleotide probes and determination of suitable conditions for hybridization are technically difficult and give cross-specificity, mak ing this method impractical for high-resolution typing (2, 9). At present, the modified PCR-RFLP method is the quickest (completion in 6—8h), requires only a small amount of template DNA, and gives highly reproducible, accurate results. Ours is the first report of high resolution genotyping of RCC patients using the modified PCR-RFLP method. The results suggest that certain HL.4-DRBJ alleles have a protective function in the development and prognosis of RCC.