Rapid HLA-DR genotyping by PCR-amplification with sequence-specific primers.

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.