title: Rapid HLA-DR genotyping by PCR-amplification with sequence-specific primers.
abstract: 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.
authors.affiliation: Renal Transplant Center, Shanghai First People's Hospital.
authors.forename: J
authors.initials: J
authors.lastname: Tan
authors.forename: T
authors.initials: T
authors.lastname: Xie
authors.forename: Q
authors.initials: Q
authors.lastname: Xu
authors.forename: D
authors.initials: D
authors.lastname: Xu
authors.forename: X
authors.initials: X
authors.lastname: Wang
mesh_terms: 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
chemical_list: D006684:HLA-DR Antigens