A sequencing-based typing method for HLA-DQA1 alleles

Abstract

Sequencing-based typing (SBT) is the most comprehensive method for characterizing human leukocyte antigen gene polymorphisms. Development of a SBT method for DQA1 is hampered because of a deletion of codon 56 in nearly half of the known DQA1 alleles. Sequence electropherograms of heterozygous samples comprising a deletion allele and a non–deletion allele display misalignment after codon 56 because of a three base-pair shift in the deletion allele. To overcome this problem, we have designed three group-specific primer sets to selectively amplify the deletion alleles from the nondeletion alleles. DNA samples are initially polymerase chain reaction (PCR)-typed using these primer sets along with an internal positive control primer set specific to growth hormone gene 1 (hGH1). The positive group-specific PCR reactions were selectively repeated without hGH1 control primers, and the amplicons were used as template in sequencing reactions. The sequence data were analyzed to obtain DQA1 types using ABI MatchTools software as well as the newly available Conexio Genomics Assign SBT Genotyping Software. The method was validated using a panel of reference DNA from the University of California, Los Angeles, International DNA Exchange Program. We conclude that the present SBT method is a technically simple and robust procedure to characterize the sequence polymorphisms in exon 2 of DQA1 gene.