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

Abstract

In this study, a new sequencing-based typing strategy for the HLA-A locus is presented which involves group-specific separate amplification of exon 2 and 3 of HLA-A alleles in a first step. Conserved HLA-A locus-specific primers of intron 1 or 3 were combined in 10 primer-mixes with group-specific primers hybridizing to the 5'- or 3'-end of exon 3 or 2 for pre-typing of the HLA-A alleles in 14 allelic groups. Maximally four overlapping short amplicons are produced under identical polymerase chain reaction (PCR) conditions with individual separate amplification of exon 2 and exon 3 of the haplotypic alleles in most heterozygous combinations. Time- and money-saving one-directional Big Dye Terminator cycle sequencing is shown to provide reliable 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 sequencing-based typing (SBT) techniques the applied typing strategy minimizes the risk of unequal amplification or of drop-outs of one of the haplotypic alleles and allows unequivocal definition of the cis/ trans linkage of polymorphic positions of the complete exon 2 and exon 3 in most heterozygous cells. This also includes detection of new alleles differing in the polymorphic template generating primer annealing sites as well as in unusual combinations of known exon 2 and 3 sequences. With 10 primer sets working under identical conditions for pre-grouping and separate amplification of the haplotypic alleles our SBT procedure also could be implemented in clinical settings of large-scale stem cell donor histocompatibility testing for fast molecular HLA-A matching.

HLA Polymorphisms in Cabo Verde and Guiné-Bissau Inferred From Sequence-Based Typing

“…All subjects were typed for HLA-A, HLA-B, and HLA-DRB1 loci. SBT of the HLA-A and -B loci was performed on exon 2 and exon 3 accordingly to Kurz et al [19] and Pozzi et al [20] with minor modifications. DNA fragments amplified by polymerase chain reaction were purified and sequenced using ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kits (Applied Biosystems, Foster City, CA) in an ABI Prism 310 Genetic Analyzer (Applied Biosystems), according to the manufacturer's instructions.…”

Resolution of AmbiguousHLAGenotyping in Korean by Multi-Group-Specific Sequence-Based Typing

“…The ambiguity in an SBT may also be resolved by a group-specific polymerase chain reaction (PCR) for the separated amplification of a single haplotype or by an SBT after haplotype-specific DNA extraction 10,11. Another approach to resolve the ambiguity from the SBT is to utilize the heterozygous ambiguity resolving primers (HARPs) for the sequencing step of an SBT.…”

Discrepancies in HLA Typing by PCR-SSOP and SBT Techniques: A Case Study

“…All subjects were typed for HLA-A and HLA-B using the high-resolution sequence-based method outlined by Kurz et al (1999) and Pozzi et al (1999), respectively, with minor modifications. HLA-DRB1 was typed through the exon 2 complete sequence.…”