【摘要】 目的:制备用于nm23-H1基因mRNA实时荧光定量PCR检测的质粒标准品。方法:通过PCR扩增出目的片断,纯化后T-A连接至pMD18-T载体,转化宿主菌E.coliDH5α,获得阳性克隆;通过PCR扩增、双酶切鉴定和测序分析,确认重组质粒完整正确;大量抽提重组质粒,测定其拷贝浓度,10倍稀释成梯度标准品,并进行荧光定量PCR检测分析。结果:nm23-H1基因目的片段成功重组至pMD18-T载体上,获得的重组质粒保持了目的片段的特异性和序列完整性。梯度浓度标准质粒的荧光定量PCR结果显示,循环阈值(C t)与起始模板量的对数值之间有着良好的线性关系。结论:成功构建了nm23-H1基因实时荧光定量PCR质粒标准品。更多还原

【Abstract】 Objective:To construct recombinant plasmid pMD18-nm23-H1/p as the standards for nm23-H1 gene detection by real-time fluorescence quantitative PCR.Methods:A 253 bp DNA segment of nm23-H1 gene cDNA was amplified by PCR and T-A cloned into pMD18-T vector,then transformed into bacterium DH5α.The recombinant plasmid DNA extracted from positive clones was identified by PCR amplification,digestion with restriction endonucleases Sal I and EcoR I,and sequence determination.The concentration of purified plasmid DNA solution was determined by analyzing its absorption at 260 nm,and then ten-fold serially diluted into 108 to 102 copy/μl.Standard quantitative curves were constructed by real-time PCR detection with the series of plasmid standards.Results:The target segment was successfully recombined into pMD18-T vector with correct sequences.The results of real-time fluorescence quantitative PCR with the series of plasmid standards showed an ideal linear relationship between the logarithmic values of initial template concentration and cycle threshold values.Conclusion:A series of recombined plasmid standards for nm23-H1 gene real-time PCR analysis have been successfully constructed.更多还原