Development and verification of PCR-fingerprinting HLA-DPB1 genotyping / 中华器官移植杂志

ABSTRACT

To accelerate the identification of HLA-DPB1 matched marrow donors from unrelated population, a very simple HLA-DPB1 genotyping method called PCR-fingerprinting (PCRF) was developed according to the theory about homoduplex and heteroduplex formation from different PCR coding strands and non-coding ones. Unlike PCR-SSCP, strict laboratory condition is not needed in the PCRF. After denaturing at 94℃ for 2 min and cooling at 37℃ for 8 min, the PCR product was separated by 8% PAGE for 5 h and polymorphism band patterns would appear when the gel staining was completed with either EB or silver staining procedure. To confirm its reliability, 21 individuals from 9 family whose DPB1 genotypes assigned by PCR-RFLP were verified. It was found that there were 8 PCRF patterns corresponding to the 9 HLA-DPB1 genotypes from the 21 cases and the same DPB1 genotypes produced identical PCRF pattern except one pair.The factors on efficient separation of heteroduplexes and homoduplexes were also discussed.