[A simplified method of HLA-DR typing with the PCR method using the genomic DNA extracted from blood-absorbed filter].
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 1 min; annealing at 55 for 1 min, extension at 72 C for 2 min) 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 all the 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.
Kabayama
H
H
2nd Department of Pediatrics, Ohashi Hospital, Toho University School of Medicine.
D000293:Adolescent; D000483:Alleles; D001483:Base Sequence; D002648:Child; D004247:DNA / Q000032:analysis; D015894:Genome, Human; D006684:HLA-DR Antigens / Q000145:classification* / Q000235:genetics; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods
D006684:HLA-DR Antigens; D004247:DNA