Rapid HLA-DRB1 genotyping by nested PCR amplification.
State of the art genotyping of HLA class II alleles with group-specific DNA amplification by the polymerase chain reaction (PCR) (1) and subsequent probing with sequence-specific oligonucleotides (2-4) is not suitable for typing cadaveric organ donors since the typing procedure takes far more than one working day. We designed specific oligonucleotide primer sets for nested PCR amplification which allowed typing for all serological HLA-DR specificities (DR1-DRw18) solely by the detection of amplified DNA in the reaction mixtures after agarose gel electrophoresis. Exon 2 of the DRB genes and a DRw52-group-specific part of DRB1 exon 2 was amplified directly from cell lysates without prior DNA extraction. The amplified DNA was subjected to a second round of amplification, which employed a set of 18 nested allele- or group-specific primer pairs. All alleles which have at least a single mismatched base at the terminal 3'-nucleotide of one primer were completely refractory to amplification. This assay is easy to perform and takes less than one working day to complete. Thus, this method may prove to be suitable for DNA typing of organ donors for prospective HLA-DR matching in renal transplantation.
Bein
G
G
Institute of Immunology and Transfusion Medicine, University of Lübeck, Medical School, FRG.
Gläser
R
R
Kirchner
H
H
D000483:Alleles; D001483:Base Sequence; D015139:Blotting, Southern; D004247:DNA / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D005091:Exons; D005784:Gene Amplification / Q000235:genetics*; D005838:Genotype; D006684:HLA-DR Antigens / Q000235:genetics*; D059810:HLA-DR Serological Subtypes; D059811:HLA-DRB1 Chains; D000949:Histocompatibility Antigens Class II / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction
D006684:HLA-DR Antigens; D059810:HLA-DR Serological Subtypes; C051104:HLA-DR52 antigen; D059811:HLA-DRB1 Chains; C060086:HLA-DRw18 antigen; D000949:Histocompatibility Antigens Class II; D015345:Oligonucleotide Probes; D004247:DNA