Complete subtyping of the HLA‐A locus by sequence‐specific amplification followed by direct sequencing or single‐strand conformation polymorphism analysis

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Abstract
A variety of reasons related to the HLA class I system has complicated the application of molecular approaches to HLA class I typing. Here we present a PCR‐based HLA‐A typing strategy considering the sequence variations of the two most polymorphic exons which allows complete subtyping of the HLA‐A locus. The method is based on a sequence‐specific amplification identifying the serologically defined HLA‐A specificities. The PCR products generated by these group‐specific primers bear the sequence information necessary for a postamplification specificity step. The primer pairs are located within one exon, either exon 2 or exon 3, which avoids amplification of polymorphic intron sequences allowing subsequent single‐strand conformation polymorphism analysis and facilitating direct sequencing. Using this method we investigated 48 cell lines and 153 clinical samples. 23 PCR reactions are performed per individual for the assignment of the serological specificities A1‐A80. The reproducibility was 100% in all cell lines and 85 clinical samples typed on two separate occasions. With the exception of 13 out of 231 possible serological combinations all homozygous and heterozygous combinations of A1‐A80 can be distinguished by specific amplification patterns. Comparing the PCR based typing results with those of serology in 12% a discrepancy was found. Solid‐phase sequencing or SSCP analysis of the group‐specific PCR fragments allowed complete subtyping of the HLA‐A locus. This strategy can identify all 48 HLA‐A alleles based on the sequence variations of the 2nd and 3rd exon. 1128 homozygous and heterozygous allele combinations are possible for the HLA‐A locus. Only 4 out of these 1128 allele combinations remained unresolved.