HLA-DQA1 genotyping by bi-directional sequencing of PCR-amplified DNA spanning exon 2

We describe a simple, reliable technique for HLA-DQA1 genotyping based on direct DNA sequencing of PCR amplified fragments from genomic DNA. The alleles of DQA1 can be divided into two subsets, with one subset demonstrating a 3 base deletion in exon 2 relative to the other. Typing heterozygous individuals who possess one allele from each sub-group can be difficult using a direct sequencing approach, since the two overlapping DNA sequences move out of phase by 3 nucleotides once the point of deletion is reached. The complete sequence is obtained by performing two separate sequencing reactions with fluorescent dye primers, coming from either end of the template. This allows all heterozygous positions in exon 2 to be unambiguously assigned.