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.


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.
Tan
J
J
Renal Transplant Center, Shanghai First People's Hospital.
Xie
T
T
Xu
Q
Q
Xu
D
D
Wang
X
X
D000483:Alleles; D005838:Genotype; D006085:Graft Survival; D006684:HLA-DR Antigens / Q000235:genetics*; D006801:Humans; D016030:Kidney Transplantation; D016133:Polymerase Chain Reaction / Q000379:methods; D011300:Preoperative Care; D012680:Sensitivity and Specificity
D006684:HLA-DR Antigens