title: 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.
abstract: 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.
authors.affiliation: Laboratoire d'Histocompatibilite, Etablissement Français du Sang-Alsace, Strasbourg, France. anne.dormoy@etss.u-strasg.fr
authors.forename: A
authors.initials: A
authors.lastname: Dormoy
authors.forename: N
authors.initials: N
authors.lastname: Froelich
authors.forename: R
authors.initials: R
authors.lastname: Leisenbach
authors.forename: B
authors.initials: B
authors.lastname: Weschler
authors.forename: J-P
authors.initials: JP
authors.lastname: Cazenave
authors.forename: M-M
authors.initials: MM
authors.lastname: Tongio
mesh_terms: D017931:DNA Primers; D006680:HLA Antigens / Q000235:genetics*; D015395:Histocompatibility Antigens Class I / Q000235:genetics; D006801:Humans; D016133:Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D006680:HLA Antigens; D015395:Histocompatibility Antigens Class I