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  • Title: Rapid HLA-DR genotyping by PCR-amplification with sequence-specific primers.
    Author: Tan J, Xie T, Xu Q, Xu D, Wang X.
    Journal: Chin Med J (Engl); 1996 Sep; 109(9):720-3. PubMed ID: 9275342.
    Abstract:
    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 performed for each sample. The amplification was accomplished by 34 cycles consisting of denaturation at 94 degrees C for 30 seconds, annealing at 60 degrees C for 50 seconds and extension at 72 degrees C 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 PCR-SSP. 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 confirmed by Southern hybridization. CONCLUSIONS: Genotyping for HLA-DR by PCR-SSP is a rapid and accurate matching technique suited for clinical application.
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