Pfu高保真酶介导的直接全血检测耳聋基因突变热点的方法建立

发布时间：2018-05-03 22:25

本文选题：耳聋 + 高保真酶　；参考：《苏州大学》2011年硕士论文

【摘要】：目的:利用全血PCR结合Pfu高保真DNA聚合酶介导的突变敏感性“分子开关”技术建立先天性耳聋4个基因10个热点突变的检测方法。方法:采集临床健康体检病人及诊断为神经性耳聋病人的血液标本,提取血液白细胞中的基因组DNA,分光光度计测定其浓度;对健康体检病人的DNA样本的耳聋基因GJB2、GJB3,SLC26 A4、线粒体DNA热点突变所在的外显子进行PCR扩增并测序,明确其基因序列,这些样本作为建立“分子开关”技术检测10个热点突变方法的标准样本;构建10个位点的野生型和突变型质粒,作为检测的野生型和突变型模板;根据该10个位点的突变特点,设计多对突变检测引物,通过实验选择产物特异性高的引物作为突变检测引物;利用Pfu高保真DNA聚合酶和3’末端硫化修饰的突变检测引物对相应的野生型和突变模板进行扩增并测序验证扩增产物;优化PCR扩增条件,提高“分子开关”对突变位点的识别能力。分别以基因组DNA和全血作为DNA模板,重复上述检测步骤,尝试建立一种简化的PCR方法。将该方法用于20例健康人及耳聋患者的热点突变检测。结果:对不同的热点突变位点设计的多对突变检测引物,通过实验筛选到特异性引物;在高保真DNA聚合酶介导的PCR反应体系中,3’末端硫化修饰突变检测引物对突变模板扩增得到产物,对正常模板无扩增产物,显示“分子开关”对耳聋基因热点突变位点的特异性识别。结论:成功应用全血PCR结合高保真DNA聚合酶介导的“分子开关”技术建立了耳聋基因10个热点突变的检测方法;“分子开关”技术是一种很有应用价值的点突变检测技术,全血PCR方法也是一种值得推广的方法。
[Abstract]:Aim: to establish a method for detecting 10 hot spot mutations in 4 genes of congenital deafness by means of whole blood PCR and Pfu high fidelity DNA polymerase mediated mutagenic "molecular switch" technique. Methods: the blood samples of patients with clinical health examination and those diagnosed as neurodeafness were collected, the genomic DNA in the blood leukocytes was extracted, and the concentration of the DNA was measured by spectrophotometer. The deafness gene GJB2GJB3OSLC26A4 was amplified by PCR and sequenced in the exon of mitochondrial DNA hot spot mutation. These samples serve as standard samples for the establishment of a "molecular switch" technique for the detection of 10 hot spot mutations; construct wild and mutant plasmids of 10 loci as templates for detection of wild and mutant types; and according to the mutation characteristics of the 10 loci, Several pairs of mutation detection primers were designed and the primers with high product specificity were selected as mutation detection primers. The corresponding wild type and mutation template were amplified by Pfu high-fidelity DNA polymerase and 3'terminal vulcanization modified mutation detection primer, and the amplified products were verified by sequencing, and the conditions of PCR amplification were optimized. To improve the ability of molecular switch to recognize mutation sites. Genomic DNA and whole blood were used as DNA templates to repeat the above steps and to establish a simplified PCR method. The method was applied to the detection of hot spot mutations in 20 healthy persons and deafness patients. Results: multiple pairs of primers were designed for different hot spot mutation sites, and specific primers were screened by experiments. In the PCR reaction system mediated by high fidelity DNA polymerase, the product was obtained by the amplification of the mutated template by using the primers for the detection of vulcanized mutagenesis at the 3'end, but no amplification product was obtained for the normal template. The results showed that the molecular switch was specific to the hot spot mutation of deafness gene. Conclusion: the whole blood PCR combined with high fidelity DNA polymerase mediated "molecular switch" technique has been successfully used to detect 10 hot spot mutations of deafness gene, and "molecular switch" technique is a valuable point mutation detection technique. The whole blood PCR method is also worth popularizing.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R764.43

【参考文献】