Olerup O, Aldener A, Fogdell A
Center for BioTechnology, Karolinska Institute, NOVUM, Huddinge, Sweden.
Tissue Antigens. 1993 Mar;41(3):119-34. doi: 10.1111/j.1399-0039.1993.tb01991.x.
In the present study PCR primers were designed for detecting all phenotypically expressed DQB1 and DQA1 allelic variability, 19 and 10 alleles, respectively, by PCR amplification with sequence-specific primers (PCR-SSP). For DQB1 typing, each sample was amplified by a first set of 14 PCR primer pairs, followed in some cases by two to six additional PCR reactions. The first 14 primer pairs allowed the identification/separation of all but a few of the recently described DQB1 alleles: DQB10504, DQB10605, DQB10606 and DQB10607 would not be identified; DQB10603 and DQB10608; and DQB10301 and DQB10304, respectively, would not be distinguished. Therefore an additional set of eight DQB1 primer pairs was used for a complete DQB1 typing, including all homozygous and heterozygous combinations. For DQA1 typing, 12 PCR reactions were performed per sample, 10 for detecting variability within the second exon and two for identifying first exon polymorphism. All homozygous and heterzoygous combinations of DQA1 alleles could be resolved by these primer pairs. In addition, four primer mixes were designed for determining codon 57 of the HLA-DQB1 gene. Thirty cell lines and 120 individuals were investigated by the DQB1 and DQA1 PCR-SSP technique, as well as with the HLA-DQ beta 57 primers. The concordance between PCR-SSP typing and assigning DQB1 and DQA1 alleles from TaqI DRB-DQA-DQB RFLP analysis was 100%. The reproducibility was 100% in 30 samples investigated on two separate occasions. Amplification patterns, investigated in 15 nuclear families, segregated according to dominant Mendelian inheritance. DQB1 and DQA1 PCR-SSP typing can be performed in 2 hours, including DNA extraction, PCR amplification and post-amplification processing. The method is technically simple and the typings are easy to interpret. The cost for typing one individual is low and is independent of the number of samples analyzed simultaneously, i.e. the technique is well-suited for routine clinical use.
在本研究中，设计了PCR引物，用于通过序列特异性引物PCR（PCR - SSP）扩增检测所有表型表达的DQB1和DQA1等位基因变异，分别为19个和10个等位基因。对于DQB1分型，每个样本先用一组14对PCR引物进行扩增，某些情况下随后再进行2至6次额外的PCR反应。前14对引物能够鉴定/区分除少数最近描述的DQB1等位基因外的所有等位基因：DQB10504、DQB10605、DQB10606和DQB10607无法鉴定；DQB10603和DQB10608以及DQB10301和DQB10304无法区分。因此，另外使用一组8对DQB1引物进行完整的DQB1分型，包括所有纯合和杂合组合。对于DQA1分型，每个样本进行12次PCR反应，其中10次用于检测第二外显子内的变异，2次用于鉴定第一外显子多态性。这些引物对能够分辨所有DQA1等位基因的纯合和杂合组合。此外，设计了四组引物混合物用于确定HLA - DQB1基因的第57密码子。通过DQB1和DQA1 PCR - SSP技术以及HLA - DQβ57引物对30个细胞系和120名个体进行了研究。PCR - SSP分型与通过TaqI DRB - DQA - DQB RFLP分析确定DQB1和DQA1等位基因之间的一致性为100%。在两个不同时间对30个样本进行检测，其重复性为100%。在15个核心家庭中研究的扩增模式按照显性孟德尔遗传方式分离。DQB1和DQA1 PCR - SSP分型可在2小时内完成，包括DNA提取、PCR扩增和扩增后处理。该方法技术简单，分型结果易于解读。对一个个体进行分型成本低，且与同时分析的样本数量无关，即该技术非常适合常规临床应用。
