SEQUENCING OF HLA CLASS-I GENES BASED ON THE CONSERVED DIVERSITY OF THE NONCODING REGIONS - SEQUENCING-BASED TYPING OF THE HLA-A GENE

We present a sequencing-based typing strategy for the HLA-A locus that is generally applicable to all HLA class I genes. Sequencing-based ty ping is the method of choice for matching in unrelated bone marrow tra nsplantation on the allelic level. We determined the noncoding sequenc es of all serological antigens and most of their subtypes and discover ed a remarkably conserved diversity characterized by polymorphic seque nce motifs. In this study we took advantage of this diversity we uncov ered in the 5' flanking region, 5' untranslated region and in the intr ons 1, 2 and 3, which was related to serological families. We establis hed 12 primer mixes for setting up a PCR-based template preparation. O ur strategy is based on the separate amplification of haplotypes and t herefore defines the cis trans linkage of polymorphic sequence motifs. This allowed individual sequencing of the haplotypes in all samples h eterozygous for the broad antigens as well as the complete analysis of the polymorphic exons 2 and 3. All templates included the 2nd intron which was used as a priming site for the gene-specific 5' and 3' unive rsal sequencing primers regardless of the amplified haplotypes. The in dependent sequencing of the haplotypes allows the application of the d ye terminator cycle sequencing technique, which is less time-consuming and less laborious than dye primer chemistry. The lack of heterozygou s positions essentially facilitates on the one hand the data analysis and on th other hand the detection of new alleles. Sequencing is only required in one direction due to the absence of peak shift problems. T he results will remain unambiguous regardless of a growing HLA sequenc e data bank since this sequencing technique defines the cis trans link age of sequence motifs in more than 95% of the cases.