Generic HLA-DRB1 gene oligotyping by a nonradioactive reverse dot-blot methodology.
HLA-DRB1 allelic specificities can be determined using SSOs annealing to their complementary PCR-amplified target DNA. To perform HLA-DR oligotyping routinely for donors and recipients of bone marrow transplantation, a "reverse" dot-blot technique has been developed that consists in the hybridization of labeled PCR-amplified target DNA to SSOs that have been first attached to nitrocellulose membranes. The 15 oligonucleotides chosen enabled the following HLA-DRB1 "generic" specificities to be defined: DR1, BON, 2, 3, 4, 11, 11 JVM, 12, 13, 13 HAG, 14, 7, 8, 9, 10. The genomic DNA was amplified by asymetric PCR with incorporation of biotinylated deoxynucleotides predominantly to generate labeled single-stranded DNA. Hybridization between specific immobilized oligoprobes and target DNA was nonradioactively detected by a colorimetric reaction using alkaline phosphatase. The reverse dot-blot methodology was successfully tested, first, for the determination of HLA-DR4 subspecificities, and then the procedure was routinely applied to the generic HLA-DR oligotyping of bone-marrow donors and recipients.
Eliaou
J F
JF
Laboratory of Immunology, Saint Eloi Hospital, CHU Montpellier, France.
Palmade
F
F
Avinens
O
O
Edouard
E
E
Ballaguer
P
P
Nicolas
J C
JC
Clot
J
J
D001483:Base Sequence; D016026:Bone Marrow Transplantation / Q000276:immunology; D004247:DNA / Q000235:genetics; D015342:DNA Probes; D005069:Evaluation Studies as Topic; D005802:Genes, MHC Class II; D006684:HLA-DR Antigens / Q000235:genetics*; D059811:HLA-DRB1 Chains; D000949:Histocompatibility Antigens Class II / Q000235:genetics*; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D008969:Molecular Sequence Data; D014019:Tissue Donors
D015342:DNA Probes; D006684:HLA-DR Antigens; D059811:HLA-DRB1 Chains; D000949:Histocompatibility Antigens Class II; D004247:DNA