Abstract
The enzymatic amplification of specific DNA sequences by the polymerase chain reaction (PCR) has provided a new approach to genetic typing of HLA-class II region specificities. However, we found the existing PCR protocols to be adequate and/or time consuming, especially when large numbers of samples need to be processed. We have devised a simplified method which alleviates these problems. Genomic DNA prepared from peripheral blood nucleated cells was facilitated using the punched-out filter paper absorbed with the peripheral blood. Second exon HLA-DRB segments of the genomic DNA were amplified by the polymerase chain reaction (PCR) (30 cycles: denaturing at 94 C for 1min: annealing at 55 for 1min, extension at 72 C for 2min) using HLA-DRB primer DRB 5'-1 (ACCGGTCGTTCITGTCCCCICAGCA) and DRB 3'-1 (CTCGCCICTGCACIGTIAAGC) designed in our laboratory. The presence of specific alleles in a PCR-amplified sample was determined by dot-blot hybridization with 32 P-labeled sequence-specific-oligonucleotides (SSOs). Quantitation of the producer is being evaluated using an automated scanner.
These newly designed primers allowed amplification of the all known expressed alleles of DRB 1, B 3, B 4 and B 5 loci. This type of reproducible and precise assay is very useful in the HLA-class II typing of large number of patients.
