以人类基因组DNA和志贺氏菌粗制裂解菌液为模板,扩增115 bp的Alu基因片段和402 bp的ipaH基因片段.通过设置氧化石墨烯(Graphene Oxide,GO)和模板的浓度为变量,分别研究了氧化石墨烯对聚合酶链式反应(Polymerase chain reaction,PCR)扩增效率和灵敏度的影响;运用多轮PCR扩增方法研究了氧化石墨烯对聚合酶链式反应扩增特异性的影响.结果显示,氧化石墨烯有效提高PCR扩增效率的范围为9-14倍,提高PCR灵敏度一个数量级以及增加多轮PCR扩增中的两轮扩增反应.从原子力显微镜(Atomic force microscope,AFM)的结果可以看出,本文制备的氧化石墨烯片层厚度主要集中在0.5-0.8 nm,少数大于1 nm,总体具有较大的比表面积.除此之外,本文还发现当氧化石墨烯浓度到达μg· μL-1后,引物二聚体与氧化石墨烯的浓度呈正比,说明氧化石墨烯优化PCR的主要影响因素是其与引物的相互作用.
Taking the human genome DNA and crude bacterial lysates of Shigella as templates,115 bp Alu gene and 402 bp ipaH gene were amplified in the presence of graphene oxide(GO).We investigated the effect of GO on the polymerase chain reaction (PCR) regarding its amplification efficiency and sensitivity by setting the concentration of GO and template as variables.And the effect of GO on the specificity of PCR was evaluated via multi-cycle PCR.The results showed that GO increased the amplification efficiency by 9-14 times,sensitivity by one order of magnitude and two rounds of amplification in multi-round PCR.From the results of atomic force microscopy (AFM),the thickness of GO film layers was mainly in 0.5-0.8 nm,and only a few more than 1 nm.The film had a large specific surface area as a whole.In addition,we also found that the concentration of the dimer template was proportional to the GO concentration when the latter was in the μg· μL-1 range,indicating that the PCR optimization was mainly caused by the interaction between GO and primer.
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