Satb2基因实时荧光定量聚合酶链反应标准品的制备及应用研究  

Preparation of Satb2 Gene Standard Plasmids for Real-time Quantitative Polymerase Chain Reaction and Its Application

作　　者：张丹方[1] 唐世杰[1] 毛小炎[1] 许金华[1] 彭立红[1] 

机构地区：[1]汕头大学医学院第二附属医院唇腭裂中心,广东汕头515041

出　　处：《汕头大学医学院学报》2008年第2期67-70,共4页Journal of Shantou University Medical College

基　　金：广东省医学科研基金资助项目(A2007431)

摘　　要：目的:制备用于Satb2基因实时荧光定量聚合酶链反应(PCR)的标准品。方法:提取胚胎上颌突组织总RNA,行逆转录反应获得第一链的cDNA,再通过PCR回收获得预期Satb2基因片段;通过T-A克隆将该片段插入pGMT质粒载体;大量提取质粒,定量后进行标准曲线的制作和实时荧光定量PCR。结果:重组质粒经PCR扩增及序列测定表明,pGMT/Satb2基因已成功克隆。结论:所构建的pGMT/Satb2基因实时荧光定量PCR检测标准品应用Taqman荧光探针技术建立的标准曲线线性关系好,灵敏度和特异性高,准确可靠,此法可作为实时荧光定量PCR检测Satb2基因的标准方法。Objective： To prepare the Satb2 gene standard plasmids for real-time quantitative PCR. Methods： The total RNA was isolated from the maxillary process of the mouse embryo, and used to synthesize the first-strand eDNA via reverse transcription. With Satb2 gene specific PCR and gel purification, the Satb2 gene fragments were obtained. The fragments were then inserted into the pGMT plasmid vector. After massive extraction and identification, the plasmid was quantified and the standard curve was established. At last, the standard curve was applied in the real-time quantitative PCR. Results： The amplified products of the recombinant plasrnid by PCR and gene sequencing confirmed that the pGMT/Satb2 gene was successfully cloned. Conclusion： The recombinant plasmid and standard curve to detect the Satb2 gene by the ＂Taqman＂ approach is good at sensitivity, specificity, quantification and linear function. It can be a standard method of real-time quantitative PCR for detection of Satb2 gene.

关 键 词：实时荧光定量聚合酶链反应 标准品 TAQMAN探针 Satb2基因 

分 类 号：R446.61[医药卫生—诊断学]