基因突变敏感性分子开关检测hiv-1耐药基因突变-detection of hiv - 1 drug-resistant gene mutations by gene mutation sensitive molecular switch.docx

Detection of HIV-1 resistance mutations by proofreading polymerase-mediated mutation-sensitive on/off switch AbstractDetection of HIV-1 resistance mutations by proofreading polymerase-mediated mutation-sensitive on/off switchAbstractObjective:To establish a PCR method for the detection of seven resistance mutations in HIV-1gene by proofreading polymerase mediated mutation sensitive on/off switch. With the use of the multiplex PCR and single PCR combining with quantitative PCR technique, seven resistance mutations can be detected by melting curve and amplification curve analysis. Whether these mutations exist or not can be used to provide the main basis for the choice of anti-HIV drugs and to guide rational drug in HIV patients.Methods:The PCR products harboring the wild sequences relevant to the drug resistance mutation gene were inserted into the pDM19-T vector for transformation into E.coli JM109 competent cells for preparing wild type vector. According to the known mutations in HIV-1 gene, mutant-specific primers were designed and synthesized by biotech company directly. To prepare wild templates, primer extension reaction was performed with low-fidelity enzyme, and products having expected sequences were cloned into T vector as template for further experiments. These newly constructed templates were confirmed by DNA sequencing. In this study, we selected the main hypermutation 7 sites to study: M41L, K65R, T69N, M184V, V75I, V82A and L90M. Two directional primer extensions were performed using mutation-sensitive on/off switch in different systems. Sequence-specific forward primers targeting wild type and mutant templates respectively were designed with 3’ terminal phosphorothioate modification. Two-directional primer extension was performed using Pfu polymerases combining with quantitative PCR technique to analyze the results.Results：Seven resistance mutations were respectively detected in different systems. When wild template was used, whereas the wild-type al