A method for typing polymorphism at the HLA-A locus using PCR amplification and immobilized oligonucleotide probes

Tissue Antigens. 1994 Sep;44(3):137-47. doi: 10.1111/j.1399-0039.1994.tb02371.x.

Abstract

We have developed a simple and rapid method for DNA typing of the HLA-A locus using PCR amplification and hybridization of the PCR product, labeled with biotinylated primers, to an array of immobilized oligonucleotide probes in a single hybridization reaction (reverse dot or line blot). A single primer set (RAP1007 and DB337) is used to specifically amplify a 990-bp fragment containing the HLA-A locus exons 1, 2, and 3 from genomic DNA. This primer set is locus-specific and amplifies all HLA-A alleles. A set of 51 sequence-specific oligonucleotide (SSO) probes, 25 for exon 2 and 26 for exon 3, was immobilized to a nylon membrane by UV-crosslinking oligonucleotide probes containing a poly-thymidine "tail" added with terminal transferase. In the line blot format, all 50 SSO probes plus a control probe are immobilized on a single nylon membrane strip. The probe array was used for typing in a hybridization reaction with DNA amplified from a variety of samples. These probes can identify 37 homozygous HLA-A alleles. In the analysis of heterozygous samples, 604 heterozygous types out of 633 (95.4%) possible heterozygous probe patterns can be detected as a unique probe reactivity pattern. A simple computer program has been developed to assign the alleles and genotypes based on the probe hybridization pattern.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • HLA-A Antigens / genetics*
  • Humans
  • Molecular Sequence Data
  • Nucleic Acid Hybridization / genetics
  • Oligonucleotide Probes / genetics*
  • Polymerase Chain Reaction / methods*
  • Polymorphism, Genetic / genetics*
  • Sequence Homology, Amino Acid

Substances

  • HLA-A Antigens
  • Oligonucleotide Probes