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.docVIP

《汕头大学医学院学报》2008年6月21卷2期 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 作者：张丹方 作者单位：汕头大学医学院第二附属医院唇腭裂中心，广东汕头515041 【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。 【关键词】 实时荧光定量聚合酶链反应 标准品 Taqman探针 Satb2基因 Preparation of Satb2 Gene Standard Plasmids for Real_time Quantitative Polymerase Chain Reaction and Its ApplicationZHANG Dan_fang，TANG Shi_jie，MAO Xiao_yan，XU Jin_hua，PENG Li_hong (Cleft Lip and Palate Treatment Center，The Second Affiliated Hospital，Shantou University Medical College，Shantou515041，China) ［Abstract］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 cDNA 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 plasmid 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. ［Key Words］real_time quantitative polymerase chain reaction；standard plasmid；Satb2 gene 缺氧、皮质激素和苯妥英等因素可致唇腭裂［1］，并在诱导其发生过程中可能影响Satb2基因(近年来新发现的一个唇腭裂基因［2］)的表达。为此，本实验通过制备Satb2基因的质粒载体标准品，制作标准曲线，进一步检测Satb2基因的表达，深入研究其在唇腭裂发生中所发挥的作用及机制。 1材料与方