HLA-DQA1 GENOTYPING BY BIDIRECTIONAL 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. Typin g heterozygous individuals who possess one allele from each sub-group can be difficult using a direct sequencing approach, since the two ove rlapping DNA sequences move out of phase by 3 nucleotides once the poi nt of deletion is reached. The complete sequence is obtained by perfor ming two separate sequencing reactions with fluorescent dye primers, c oming from either end of the template. This allows all heterozygous po sitions in exon 2 to be unambiguously assigned.