A method for typing polymorphism at the HLA-A locus using PCR amplification and immobilized oligonucleotide probes.
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.
Human Genetic Department, Roche Molecular Systems, Alameda, CA.
T L
TL
Bugawan
R
R
Apple
H A
HA
Erlich
D000595:Amino Acid Sequence; D001483:Base Sequence; D015234:HLA-A Antigens / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000235:genetics; D015345:Oligonucleotide Probes / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
D015234:HLA-A Antigens; D015345:Oligonucleotide Probes