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Enhancement of LONG PCR Efficiency by Nanomaterials

Author: ZhaoZuo
Tutor: ZhangZhiZhou
School: Harbin Institute of Technology
Course: Genetics
Keywords: long PCR nanomaterials nucleus micro-molecules real time PCR
CLC: TB383.1
Type: Master's thesis
Year: 2011
Downloads: 53
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Abstract


In recent decades, there didn’t have any major breakthrough for the Long PCR technology at home and abroad until the emergence of nanotechnology. Nano- materials which as a novel PCR additives had been accepted by more and more laboratories. These nano-materials can influence the structure of some biological macromolecular by its natural capacity of surface and quantum size effect. This kind of new Long PCR enhancer provides a new perspective for the improvement of gene amplification technology.In this study, 76 kinds of carbon nano-materials and 9 kinds of metal nano- materials had been selected by the 10×buffer system for Long PCR model. In addition, 4 kinds of normal long-PCR buffer had been optimized by the ABC buffer with another 3 kinds of nucleus chemicals and their combinations. All of these PCR ingredients were all independent research and developed by our own laboratory. At the same time, another Long PCR model had been set up to test these 76 kinds of carbon nano-materials and 9 kinds of metal nano- material again. Enventually, all of the nano- materials had been detected by the real-time–Q-PCR.The results demonstrated that excepting silver nanoparticles another 8 kinds of metal nano-materials can promoted the PCR reaction obviously with their optimal concentration of 1mg/ml and 10mg/ml in the condition of the 10×buffer and Barnes9.1 system. However, other kinds of carbon nano-materials can only amplified in the condition of Barners 9.1ABD system. The concentration for the No-7, 18, 23 and 29 were 10mg/ml.ABC buffer can significantly optimized the Tricine buffer and Bicine buffer and 3 kinds of nucleus chemicals with their combination can promted the Barners 9.1 obviously. Moreover, in the following test, Barners 9.1 had been proved a remarkably effects for the 20kb aiming fragments, even for 30kb.In the Q-PCR test, carbon nano-materials with the concentration of 10mg/ml and 1mg/ml had a serious quenches for the fluorescence. But there didn’t any effects for the metal nano-materials. Different nano-materials with their different concentration varied their Tm values dependently. The following nano-materials can deceased the Ct values obviously and can improve amplification efficiency. They are No-47 with the concentration of 1mg/ml and No-1、2、8、25、27、28、30、33、47、48、49、55、61、66 with the concentration of 0.1mg/ml, Colorless Ag with the concentration of 10mg/ml. In the subject, 4 kinds of nano-particles and 8 kinds of metal nano-materials had been selected. This can pave the way the mechanism of the following research. By the way, the combination of ABD with the Barnes 9.1 buffer had been made to the PCR kit and input the market. This must promoted the development of Long PCR technology to some extent.

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