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 typing is the method of choice for matching in unrelated bone marrow transplantation on the allelic level. We determined the noncoding sequences of all serological antigens and most of their subtypes and discovered a remarkably conserved diversity characterized by polymorphic sequence motifs. In this study we took advantage of this diversity we uncovered in the 5' flanking region, 5' untranslated region and in the introns 1, 2 and 3, which was related to serological families. We established 12 primer mixes for setting up a PCR-based template preparation. Our strategy is based on the separate amplification of haplotypes and therefore defines the cis/trans linkage of polymorphic sequence motifs. This allowed individual sequencing of the haplotypes in all samples heterozygous 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' universal sequencing primers regardless of the amplified haplotypes. The independent sequencing of the haplotypes allows the application of the dye terminator cycle sequencing technique, which is less time-consuming and less-laborious than dye primer chemistry. The lack of heterozygous positions essentially facilitates on the one hand the data analysis and on the other hand the detection of new alleles. Sequencing is only required in one direction due to the absence of peak shift problems. The results will remain unambiguous regardless of a growing HLA sequence data bank since this sequencing technique defines the cis/trans linkage of sequence motifs in more than 95% of the cases.
Kotsch
K
K
Department of Internal Medicine, Bloodbank, Virchow-Klinikum, Humboldt University, Berlin, Germany.
Wehling
J
J
Köhler
S
S
Blasczyk
R
R
D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D005805:Genes, MHC Class I; D015234:HLA-A Antigens / Q000145:classification / Q000235:genetics*; D006239:Haplotypes; D015395:Histocompatibility Antigens Class I / Q000145:classification / Q000235:genetics*; D006801:Humans; D007438:Introns / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D015234:HLA-A Antigens; D015395:Histocompatibility Antigens Class I