Generic HLA-DRB1 gene oligotyping by a nonradioactive reverse dot-blot methodology

Hum Immunol. 1992 Dec;35(4):215-22. doi: 10.1016/0198-8859(92)90002-5.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Bone Marrow Transplantation / immunology
  • DNA / genetics
  • DNA Probes
  • Evaluation Studies as Topic
  • Genes, MHC Class II
  • HLA-DR Antigens / genetics*
  • HLA-DRB1 Chains
  • Histocompatibility Antigens Class II / genetics*
  • Histocompatibility Testing / methods*
  • Humans
  • Immunoblotting / methods*
  • Molecular Sequence Data
  • Tissue Donors


  • DNA Probes
  • HLA-DR Antigens
  • HLA-DRB1 Chains
  • Histocompatibility Antigens Class II
  • DNA