Mono-allelic amplification of exons 2-4 using allele group-specific primers for sequence-based typing (SBT) of the HLA-A, -B and -C genes: Preparation and validation of ready-to-use pre-SBT mini-kits

• Published in: Tissue antigens 2003-09, Vol.62 (3), p.201-216
• Main Authors: Dormoy, A., Froelich, N., Leisenbach, R., Weschler, B., Cazenave, J-P., Tongio, M-M.
• Format: Article
• Language: English
• Subjects: DNA Primers; high-resolution DNA typing; Histocompatibility Antigens Class I - genetics; HLA Antigens - genetics; HLA class I; Humans; mono-allelic SBT; Polymerase Chain Reaction
• ISSN: 0001-2815; 1399-0039
• DOI: 10.1034/j.1399-0039.2003.00035.x

Class I allelic typing based on sequencing is reliable, immutable and easy to analyse when only one allele is amplified using a specific mono‐allelic technique. A strategy has been developed to selectively amplify exons 2, 3 and 4 of each allele of the three class I loci, previously identified by generic typing, in order to sequence these alleles from their intronic parts in only one direction. This procedure is based mainly on the polymorphism of exon 1 and intron 1 of the HLA‐A, ‐B and ‐C genes with allele group‐specific forward primers and locus‐specific reverse primers so as to perform mono‐allelic amplification in a ‘One Step’ pre‐sequence‐based typing (pre‐SBT) PCR. The 5′ polymorphism found at each locus is nevertheless not sufficient to discriminate all allelic combinations. Hence exon 2 and exon 3 polymorphism had to be used in a ‘Two Step’ pre‐SBT PCR method to selectively amplify the two alleles in the 1.8%, 7.6% and 0.9% of unresolved combinations found in our laboratory for, respectively, the HLA‐A, ‐B and ‐C loci. Preparation and validation of ‘ready‐to‐use’ aliquots of primer‐mixes, pre‐SBT buffer and sets of Dye terminator reaction mixtures containing locus‐specific intronic primers makes the procedure easy and efficient. The SBT method is the only allelic typing technique used in our laboratory (to date, 742 HLA‐A*, 802 HLA‐B* and 615 HLA‐Cw* alleles have been sequenced) and our successful participation in the national and international quality controls of 4 years ago testifies to the accuracy of the results.