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
B. Kurz et al., 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, TISSUE ANTI, 53(1), 1999, pp. 81-96
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.