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Objective To establish a rapid genotyping for HLA-DR alleles by polymerase chain reaction with sequence-specific primers(PCR-SSP)for clinical application. Material and Methods The subjects of study included 69 recipients, 43 unrelated donors and 5 cell lines, Genomic DNA was prepared from peripheral blood leukocytes by a salting-out method. Thirty primers were designed according to the HLA-DRB nucleotide sequences, and synthesized on a 391 DNA synthesizer, Twenty separate PCR reactions were perfomed for each sample, The amplification was accomplished by 34 cycles consisting of denaturation at 94℃ for 30 seconds, annealing at 60℃ for 50 seconds and extension at 72℃ for 40 seconds. The specificity of matching was determined by standard DNAs and Southern hybridization using DIG labeling probes. Results All 112 samples and 5 cell lines were able to be typed by PCRSSP. No false positive or false negative typing results were obtained. The reproducibility was 100%. The size of the specific product was in concordance with the size of the designed primers. The overall time for genotyping was 4 hours. The typing results were confimed by Southem hybridization. Conclusions Genotyping for HA-DR by PCR-SSP is a rapid and accurate matching technique suited for clinical application.
Objective To establish a rapid genotyping for HLA-DR alleles by polymerase chain reaction with sequence-specific primers (PCR-SSP) for clinical application. Material and Methods The subjects of study included 69 recipients, 43 unrelated donors and 5 cell lines, Genomic DNA was prepared from peripheral blood leukocytes by a salting-out method. Thirty primers were designed according to the HLA-DRB nucleotide sequences, and synthesized on a 391 DNA synthesizer, Twenty separate PCR reactions were perfomed for each sample, The specificity of matching was determined by standard DNAs and Southern hybridization using DIG labeling probes. Results All 112 samples and 5 cell lines were able to be typed by PCRSSP. No false positive or false negative typing results were obtained. The reproducibility was 100%. The size of the speci The overall results for genotyping was 4 hours. The typing results were confimed by Southem hybridization. Conclusions Genotyping for HA-DR by PCR-SSP is a rapid and accurate matching technique suited for clinical application.