Sequence-based typing of HLA-B: the B7 cross-reacting group

The large number of polymorphic sites in the HLA-B locus makes sequencing a n efficient way of detecting and analysing them. Most polymorphic sites are located in the alpha1 and alpha2 domains of the molecule, encoded by exons 2 and 3 of the gene. An HLA-B-specific sequence-based typing (SBT) strateg y was designed for routine application identifying the polymorphic sites in these domains. Exons 2 and 3 were amplified separately using amplification primers located in intron 1, intron 2 and intron 3. Separate amplification of exons 2 and 3 resulted in short polymerase chain reacting (PCR) product s and enabled a solid-phase sequencing approach, which made correct assignm ent of heterozygous positions possible due to low background. A one-step se quencing reaction was performed using fluorescent dye-labelled sequencing p rimers. One forward sequencing reaction was performed for exon 2, whereas f or exon 3, two forward sequencing reactions were needed using two different sequencing primers located in intron 2 and exon 3. The combined sequences of exon 2 and 3 were used for automatic alignment to an HLA-B sequence data base and automatic allele assignment. A total of 355 individuals with at le ast one allele belonging to the B7 cross-reacting group (B7, 13, 22, 27, 40 , 41, 42, 47, 48, 81 and 82) were typed for HLA-B by SET. In the B7 group 4 8 different alleles were identified, in the non-B7 group a further 59 allel es were sequenced, 9 new alleles were identified. The sequencing strategy d escribed has proven to be reliable and efficient for high-resolution HLA-B typing.