Arguello R, Avakian H, Goldman J M, Madrigal J A
Anthony Nolan Research Institute, London, United Kingdom.
Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):10961-5. doi: 10.1073/pnas.93.20.10961.
We describe a novel high resolution DNA based typing approach for HLA class I alleles, which identifies the recombinational motifs present in exons 2 and 3 of the HLA class I genes. Unique identification patterns for 201 known HLA-A, HLA-B, and HLA-Cw alleles were generated by the use of only 40 probes, which were targeted at these common motifs. The unambiguous identification of the alleles was achieved by the development of a new and powerful allelic separation technique that allows isolation of single alleles after amplification. To validate the method, we have used locus-specific primers to amplify exons 2 and 3 of HLA-A, HLA-B, and HLA-Cw loci from 22 heterozygous and 41 homozygous cell lines. After amplification, the allelic fragments from each locus were separated, blotted, and hybridized with the 40 probes. In all cases, the allelic products could be separated and 81 different class I alleles, 33 HLA-A, 30 HLA-B, and 18 HLA-Cw, were identified according to the predicted probe hybridization patterns.
我们描述了一种用于HLA I类等位基因分型的新型高分辨率DNA方法，该方法可识别HLA I类基因外显子2和3中存在的重组基序。仅使用40个针对这些常见基序的探针，就生成了201种已知HLA - A、HLA - B和HLA - Cw等位基因的独特识别模式。通过开发一种新的强大等位基因分离技术实现了等位基因的明确识别，该技术允许在扩增后分离单个等位基因。为验证该方法，我们使用位点特异性引物从22个杂合细胞系和41个纯合细胞系中扩增HLA - A、HLA - B和HLA - Cw位点的外显子2和3。扩增后，分离每个位点的等位基因片段，进行印迹，并与40个探针杂交。在所有情况下，等位基因产物均可分离，并根据预测的探针杂交模式鉴定出81种不同的I类等位基因，其中33种HLA - A、30种HLA - B和18种HLA - Cw。
