磁富集多重PCR扩增技术及其应用

发布时间：2018-05-07 06:08

  本文选题：γ-Fe_2O_3 + 杂交　； 参考：《湖南工业大学》2012年硕士论文

【摘要】：聚合酶链式反应（polymerase chain reaction, PCR）由于其特异性强、灵敏度高、快速简便等优点，一直是生命科学领域一种重要的研究手段。增强其特异性，提高其灵敏度，是PCR技术研究的重点。 我们制备了平均粒径为15nm的γ-Fe_2O_3磁性纳米粒子，并对其进行链霉亲和素修饰。实验证明链霉亲和素修饰的γ-Fe_2O_3磁性纳米粒子具有良好的生物活性，可以进一步用于生物检测。 我们将磁性纳米粒子γ-Fe_2O_3和多重PCR技术相结合，进行了磁富集多重PCR扩增，以增强其特异性，提高检测灵敏度。 首先我们对该方法的可行性进行分析，进行了磁富集靶序列单重PCR扩增。在这一实验过程中，先将生物素修饰的特异性引物和靶序列杂交，然后通过链霉亲和素修饰的磁性纳米粒子γ-Fe_2O_3将其富集到表面，通过变性获取单链靶序列，再使用目的序列的扩增引物进行PCR扩增。通过对实验条件的优化，，我们得到靶序列和特异性引物杂交的最适温度为52.5℃，链霉亲和素修饰的磁性纳米粒子γ-Fe_2O_3的最佳用量为90μg，该方法对靶序列检测的灵敏度为5×10-10ng/μL。确定了该方法的可行性。 然后我们设计了AGT基因M235T位点和A-6G位点，MTHFR基因A1298C位点和C677T位点的扩增引物，在磁富集单重PCR的基础之上，对这四个位点进行磁富集多重PCR扩增，并优化了扩增条件。通过优化实验我们得到，这四个位点的磁富集多重PCR的退火温度为56℃，在30μL扩增体系中，25mmol/L的Mg2+用量为2.0μL，10μmol/L的dNTP用量为1.5μL，5U/μL的Taq酶用量为0.7μL，10mmol/L的四个位点引物的用量分别为：C677T1μL、M235T0.7μL、A1298C1μL、A-6G1.5μL。同时得到这四个位点的磁富集多重PCR扩增的灵敏度为5×10-9ng/μL。 最后，结合通用引物PCR技术和基因芯片技术，对磁富集多重PCR扩增方法在这四个位点的SNP分型上进行了初步应用研究。
[Abstract]:Polymerase chain reaction (PCR) has been an important research method in the field of life science because of its high specificity, high sensitivity, rapidity and simplicity. Enhancing its specificity and sensitivity is the focus of PCR technology. 纬 -Fe _ 2O _ 3 magnetic nanoparticles with average diameter of 15nm were prepared and modified by streptavidin. The results show that the 纬 -Fe _ 2O _ 3 magnetic nanoparticles modified by Streptomycin have good biological activity and can be used for biological detection. The magnetic nanoparticles 纬 -Fe2O3 and multiplex PCR were combined to amplify PCR with magnetic enrichment to enhance its specificity and detection sensitivity. Firstly, we analyzed the feasibility of this method, and carried out single PCR amplification of magnetic enrichment target sequence. In this experiment, the specific primers and target sequences modified by biotin were first hybridized and then enriched onto the surface by the magnetic nanoparticles 纬 -Fe2O3 modified by streptavidin, and the single-chain target sequences were obtained by denaturation. The primers of the target sequence were used for PCR amplification. By optimizing the experimental conditions, the optimum temperature of target sequence and specific primer hybridization was 52.5 鈩

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