HLA-DQB1 AND HLA-DQA1 TYPING BY PCR AMPLIFICATION WITH SEQUENCE-SPECIFIC PRIMERS (PCR-SSP) IN 2 HOURS

In the present study PCR primers were designed for detecting all pheno typically expressed DQB1 and DQA1 allelic variability, 19 and 10 allel es, respectively, by PCR amplification with sequence-specific primers (PCR-SSP). For DQB1 typing, each sample was amplified by a first set o f 14 PCR primer pairs, followed in some cases by two to six additional PCR reactions. The first 14 primer pairs allowed the identification/s eparation of all but a few of the recently described DQB1 alleles: DQB 10504, DQB1*0605, DQB1*0606 and DQB1*0607 would not be identified; DQ B10603 and DQB1*0608; and DQB1*0301 and DQB1*0304, respectively, woul d not be distinguished. Therefore an additional set of eight DQB1 prim er pairs was used for a complete DQB1 typing, including all homozygous and heterozygous combinations. For DQA1 typing, 12 PCR reactions were performed per sample, 10 for detecting variability within the second exon and two for identifying first exon polymorphism. All homozygous a nd heterozygous combinations of DQA1 alleles could be resolved by thes e primer pairs. In addition, four primer mixes were designed for deter mining codon 57 of the HLA-DQB1 gene. Thirty cell lines and 120 indivi duals were investigated by the DQB1 and DQA1 PCR-SSP technique, as wel l as with the HLA-DQbeta57 primers. The concordance between PCR-SSP ty ping and assigning DQB1 and DQA1 alleles from TaqI DRB-DQA-DQB RFLP an alysis was 100%. The reproducibility was 100% in 30 samples investigat ed on two separate occasions. Amplification patterns, investigated in 15 nuclear families, segregated according to dominant Mendelian inheri tance. DQB1 and DQA1 PCR-SSP typing can be performed in 2 hours, inclu ding DNA extraction, PCR amplification and post-amplification processi ng. The method is technically simple and the typings are easy to inter pret. The cost for typing one individual is low and is independent of the number of samples analyzed simultaneously, i.e. the technique is w ell-suited for routine clinical use.