Rasmussen Henrik, Werge Thomas
Research Institute of Biological Psychiatry, H:S Sct. Hans Hospital, 2 Boserupvej, 4000 Roskilde, Denmark.
Mol Cell Probes. 2007 Feb;21(1):8-11. doi: 10.1016/j.mcp.2006.05.003. Epub 2006 May 27.
We have developed a closed-tube assay for determination of the chemokine receptor type 5 (CCR5) 32-bp deletion allele, which protects against infections with HIV and modulates susceptibility to a variety of inflammatory diseases. This assay utilizes dissociation analysis of amplified products in the presence of Sybr Green I for allele discrimination. After having established robust conditions for the assay, we used it to genotype 590 unknown DNA samples. A blinded comparison with a procedure based upon agarose gel electrophoresis of amplified material revealed complete concordance between the two procedures. Our closed-tube assay is inexpensive and easy to carry out. Furthermore, it reduces or eliminates the risk of carry-over contamination with previously amplified products. The insights gained in this study can be applied to develop assays for genotyping of other insertion/deletion polymorphisms based upon differences in T(m) of allele-specific amplicons.
我们开发了一种用于检测趋化因子受体5（CCR5）32碱基对缺失等位基因的闭管检测法，该等位基因可预防HIV感染并调节对多种炎症性疾病的易感性。此检测法利用在SYBR Green I存在下对扩增产物进行解离分析来鉴别等位基因。在为该检测法建立了稳定的条件后，我们用它对590个未知DNA样本进行基因分型。与基于扩增材料琼脂糖凝胶电泳的方法进行的盲法比较显示，两种方法结果完全一致。我们的闭管检测法成本低廉且易于操作。此外，它降低或消除了被先前扩增产物污染的风险。本研究中获得的见解可应用于开发基于等位基因特异性扩增子熔解温度（Tm）差异的其他插入/缺失多态性基因分型检测法。
