G-四链体级联信号扩增可视化在转基因检测中的应用

发布时间：2018-03-01 22:15

  本文关键词： G-四链体 级联信号扩增 可视化 转基因检测　出处：《合肥工业大学》2017年硕士论文　论文类型：学位论文

【摘要】：伴随着生物技术的进步,转基因作物在全球范围内得到广泛的种植。转基因技术打破了生殖隔离的限制,使得转基因作物整合了多物种的基因。虽然未有科学数据直接证明,但是转入的外源基因仍然对环境和食品安全构成潜在的威胁。所以对于转基因作物以及相关产品的安全性评价、监管和检测显得尤为重要。本论文主要研究利用新型核苷酸探针和DNA扩增方法联用对转基因成分进行检测的方法。在研究过程中,使用核苷酸探针与DNA扩增方法联合使用,并探索出合适的体系,以及联用体系在不同条件下的通用性。通过设计一段包含G-四链体互补序列的特异性探针用于等温扩增方法,对含Bt基因部分序列的单链DNA分子进行可视化检测。通过优化,确定了1μL 10X Isothermal Amplification和0.5μL 10X NEBuffer 3.1、10mM Mg~(2+)、4μM Hemin、5U Nb.BbvCI、4U Bst 2.0 WarmStar,并使用三片层的分子内G-四链体探针作为最优反应体系,孵育60min完成对目的基因的检测。建立了一种简单的,无需标记的G-四链体等温扩增检测体系。进一步通过将G-四链体核苷酸探针和两种DNA扩增方法联用。以转基因水稻科丰6号基因组DNA作为对cry1Ab/cry1Ac基因检测的目标DNA,为了降低背景信号对检测结果的影响,使用初始1μM浓度的含有分子内G-四链体结构的引物进行PCR反应。当体系内存在存在cry1Ab/cry1Ac基因的植物基因组DNA时,会通过PCR和等温扩增的反应过程,使体系内积累大量的G-四链体,并通过显色系统使本来无色的溶液变成蓝绿色。基于此,建立了一种可以直接从转基因实际样品中检测cry1Ab/cry1Ac基因的可视化检测方法。除此之外,还探索了级联扩增方法在检测其他转基因作物样品时,特别是具有复杂基因组结构的转基因玉米MON89034的检测效果。使用5μL和2μL的对应各自检测体统的引物分别用于Nb.BbvCI和Nt.BstNBI检测体系的PCR过程,之后再分别在37℃和55℃酶的最适温度下孵育30min。最终Nb.BbvCI体系和Nt.BstNBI体系分别对MON89034的检出限为100拷贝和50拷贝,达到了转基因检测的要求。因此,建立了一种选择性好,并且具有通用性的可视化转基因检测方法。
[Abstract]:With advances in biotechnology, genetically modified crops have been widely planted worldwide. Transgenic technologies have broken the restrictions of reproductive isolation and led to the integration of multi-species genes into genetically modified crops, although there is no direct scientific data to prove it. But the foreign genes still pose a potential threat to the environment and food safety. In this paper, we mainly study the method of detecting transgenic components by using new nucleotide probe and DNA amplification method. In the process of research, we use nucleotide probe and DNA amplification method. The suitable system and the versatility of the combined system under different conditions were explored. A specific probe containing the complementary sequence of G-quadruplex was designed for isothermal amplification. Visual detection of single-stranded DNA molecule containing partial sequence of BT gene was carried out. Through optimization, 1 渭 L 10X Isothermal Amplification and 0.5 渭 L 10X NEBuffer 3.1mM Mg~(2 were determined as 4 渭 M Hemin5U NB. BbvCI4U Bst 2.0 WarmStar.Three layers of intramolecular G-quad probe were used as the optimal reaction system. After incubation for 60 minutes, the target gene was detected. The G-quad nucleotide probe and two DNA amplification methods were further used to detect the cry1Ab/cry1Ac gene in transgenic rice Kefeng 6 genomic DNA. In order to reduce the influence of background signal on the detection results, The PCR reaction was carried out by using primer containing intramolecular G- quadruplex structure with initial concentration of 1 渭 M. When there was plant genomic DNA with cry1Ab/cry1Ac gene in the system, it would accumulate a large number of G- quartiles through the reaction process of PCR and isothermal amplification. Based on this, a visualized method for detecting cry1Ab/cry1Ac gene directly from actual transgenic samples was established. The cascaded amplification method was also explored in the detection of other transgenic crop samples, In particular, the detection effect of transgenic maize MON89034 with complex genomic structure was studied. 5 渭 L and 2 渭 L primers were used to detect the PCR process of Nb.BbvCI and Nt.BstNBI detection system, respectively. Then incubated at the optimum temperature of 37 鈩，

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