高保真DNA聚合酶分子开关在EGFR基因突变检测中的应用-药理学专业论文.docxVIP

 PAGE 4 III 中文提要 高保真 DNA 聚合酶分子开关在 EGFR 基因突变检测中的应用 突变患者两例，经测序验证结果正确，表明该检测平台完全可以应用到临床诊断。 结论：高保真酶介导的突变敏感性分子开关技术，对 EGFR 基因突变的检测有较 高的特异性和灵敏度，其高灵敏性的优点对微量突变的检测有重大的临床意义，优于 传统测序技术，是指导临床个体化用药，产前诊断以及肿瘤早期发现的有力的分子诊 断工具。 关键词：高保真酶分子开关；基因诊断； 肺癌； EGFR 基因 作 者：刘 彬 指导教师：李 凯 A Mutation Sensitive Switch Assay to Detect Five Clinically Significant EGFR Mutations Abstract A Mutation Sensitive Switch Assay to Detect Five Clinically Significant EGFR Mutations Abstract Objective: To develop methods to detect five common EGFR somatic mutations in tumor tissues from NSCLC patients by using a nanoscale mutation-sensitive switch consisting of a high fidelity polymerase and phosphorothioate-modified allele-specific primers. Methods: Mutant templates and wild type templates were obtained from either regular PCR using Taq DNA polymerase or overlap extension PCR. The PCR products were gel purified and sub-cloned into the PMD19-T vector. Cloning products harboring both normal and mutant EGFR fragments were confirmed by direct sequencing. Blood samples were obtained from normal controls, and tissue samples were from lung cancer patients. The five clinically significant EGFR mutations examined here are: S768I, T790M, L858R and 15-bp and 18-bp deletion mutations. Five pairs of primers were used to detect the five mutations. The forward primers had a 3 terminal phosphorothioate modification while the common reverse primer had no specific modification. A nanoscale proofreading polymerase-mediated on/off switch was employed in these five loci to detect the S768I, T790M and L858R point mutations as well as the 15-bp (E746-A750) and 18-bp (L747–S752) deletion mutants in exon 19. The molarity of the wild type and mutant templates was adjusted to 1:1 and then diluted to 107, 106, 105, 104, 103, 102 and 101 copies/μL to test the sensitivity of the phosphorothioate modification and high fidelity enzyme. Rear-time PCR and multiplex PCR were also used in this assay. Results: The wild typ