New high resolution typing strategy for HLA-A locus alleles based on dye terminator sequencing of haplotypic group-specific PCR-amplicons of exon 2 and exon 3

In this study, a new sequencing-based typing strategy for the HLA-A locus i s presented which involves group-specific separate amplification of exon 2 and 3 oi HLA-A alleles in a first step. Conserved HLA-A locus-specific prim ers oi intron I or 3 were combined in 10 primer-mixes with group-specific p rimers hybridizing to the 5'- or 3'-end of exon 3 or 2 for pre-typing of th e HLA-A alleles in 14 allelic groups. Maximally four overlapping short ampl icons are produced under identical polymerase chain reaction (PCR) conditio ns with individual separate amplification of exon 2 and exon 3 of the haplo typic alleles in most heterozygous combinations. Time- and money-saving one -directional Big Dye Terminator cycle sequencing is shown to provide reliab le high resolution typing of the HLA-A alleles, even in a few cases of two amplicons in one primer reaction mixture. In comparison, to other sequencin g-based typing (SBT) techniques the applied typing strategy minimizes the r isk of unequal amplification or of drop-outs of one of the haplotypic allel es and allows unequivocal definition of the cis/trans linkage of polymorphi c positions of the complete exon 2 and exon 3 in most heterozygous cells. T his also includes detection of new alleles differing in the polymorphic tem plate generating primer annealing sites as well as in unusual combinations of known exon 2 and 3 sequences. With 10 primer sets working under identica l conditions for pre-grouping and separate amplification of the haplotypic alleles our SET procedure also could be implemented in clinical settings of large-scale stem cell donor histocompatibility testing for fast molecular HLA-A matching.