Haras D, Piperi M H, Cucchi-Mouillot P
Université de Corse, faculté des sciences et techniques, laboratoire de biologie moléculaire appliquée, Corte, France.
C R Acad Sci III. 1995 Sep;318(9):977-84.
Since 1989, several HLA-DQA1 PCR-RFLP genotyping protocols have been published. These methods require complete digestion of the PCR products and determination of the restriction fragments length. The HLA-DQA1 PCR-RFLP genotyping protocol describe here uses one amplification step through PCR, digestion of the PCR-products with 8 restriction endonucleases, and determination of the fragments size after polyacrylamide gel electrophoresis. Five of the enzymes, having no more than one restriction site in each allele (ApaLI, HphI, BsaJI, FokI and MboII), allow distribution of all the genotypes in 19 allelic-combination groups on the base of the digestion pattern: cut/not cut. Three additional enzymes (MnlI, DdeI and RsaI), having at least 2 restriction sites in each allele, are used to assign the genotype in each allelic-combination group on the base of the restriction fragments length observed. Eight of the 13 alleles, 36 of the 91 HLA-DQA1 genotypes could be characterized. Four to 8 samples could be characterized each day, including DNA extraction. The number of endonucleases used could act as internal control of enzymatic activities and the genotyping protocol can include new HLA-DQA1 alleles without modification of the experimental steps. This protocol can be applied easily in a laboratory without specific technical training or specific equipment.
自1989年以来，已发表了几种HLA - DQA1 PCR - RFLP基因分型方案。这些方法需要对PCR产物进行完全消化并测定限制性片段长度。本文所述的HLA - DQA1 PCR - RFLP基因分型方案通过PCR进行一步扩增，用8种限制性内切酶消化PCR产物，并在聚丙烯酰胺凝胶电泳后测定片段大小。其中5种酶在每个等位基因中不超过一个限制性位点（ApaLI、HphI、BsaJI、FokI和MboII），根据消化模式（切割/未切割）可将所有基因型分为19个等位基因组合组。另外3种酶（MnlI、DdeI和RsaI）在每个等位基因中至少有2个限制性位点，用于根据观察到的限制性片段长度在每个等位基因组合组中确定基因型。13个等位基因中的8个，91种HLA - DQA1基因型中的36种可以被鉴定。包括DNA提取在内，每天可鉴定4至8个样本。所用内切酶的数量可作为酶活性的内部对照，并且该基因分型方案可以在不修改实验步骤的情况下纳入新的HLA - DQA1等位基因。该方案无需特定技术培训或特定设备即可在实验室中轻松应用。
