Pyrosequence-based typing of alleles of the HLA-DQB1 gene.

DNA typing of alleles of the highly polymorphic HLA-DQBI gene was performed by Pyrosequencing using purified DNA from the 11th International Histocompatibility Workshop human cell lines and samples from the Children's Hospital of Pittsburgh registry of diabetics and their first-degree relatives. Pyrosequencing was optimized for genotyping exon 2 of the HLA-DQB1 gene, but the procedure should be applicable to other HLA loci. The 47 HLA-DQB1 alleles were readily identifiable, as were the 1,128 potential allelic heterozygous combinations. The method required PCR conditions that specifically amplified DQB1 but not the pseudogene, DQB2. The new method of pyrosequence-based typing can be performed in 96- or 384-well format. The 61 polymorphic residues of DQB1 exon 2 were identified within four pyrosequencing reactions, obtained by a 70-nucleotide read length in each reaction, in about an hour's time. Allelic combinations of HLA-DQB1 most frequentlyfound in the population of diabetics and their immediate family members were analyzed and successfully compared to typing of the DQB1 alleles by sequence-specific oligonucleotide probe protocols. Pyrosequence-based typing is compatible with genotyping of allelic combinations expected from heterozygous individuals, resulting in nucleotide resolution of the highly polymorphic HLA system. Using pyrosequencing, more than 750 sample wells can be processed in a working day, resulting in the identification of more than 50,000 bases.