The Mechanism Study Of Polyethyleneimine-Based Derivatives And Nanocomposites On PCR Enhancing Effect

Polymerase Chain Reaction (PCR) is an in vitro DNA amplification technology. PCR has been one of the most common and important molecular biological techniques. PCR technology can now be applied to sequencing, pathogen detection, genetic analysis and other fields. PCR reaction mechanism is very complicated and PCR could be affected by a number of factors in practice. This technology could face several "bottlenecks", e.g. the low yield, low sensitivity, the low specificity, etc. Improvements of the specificity and efficiency of PCR are always required. It is proven that by adding additives, some non-specific amplification problems could be solved to certain extent. Finding new additives to enhance the specificity and efficiency of PCR is still valuable and the understanding of the PCR enhancing mechanism still remains a great challenge.In this study, we found a new enhancer:polyethylene (PEI) which could enhance the specificity of the PCR, and based on this phenomenon, we researched the additive enhancing mechanism on PCR.We established two PCR systems to test the role of enhancer in PCR. One system is a two-round unspecific system which used Pseudomonas aeruginosa genome as a template; the other system is a 14.385kb product long PCR system usingλDNA as a template. The two systems reacted with all kinds of additives in our study and the specificity has been enhanced.There are several reported enhancing mechanisms of additives on PCR reaction. One about MWCNT that can optimize the PCR based on its good thermal conductivity. In our study, we found that PEI modified MWCNT, MWCNT-PEI has the same optimization result to MWCNT, but its optimum concentration is less. Considering the cationic nature of PEI, our laboratory synthesized positively charged partially (50%) acetylated PEI (PEI-Ac50), completely acetylated PEI (PEI-Ac) and other PEI derivatives for comparison with PEI. We show that they are able to improve the PCR specificity with an optimum concentration order of PEI< PEI-Ac50< PEI-Ac, whereas negatively charged carboxyl-terminated PEI (PEI-SAH) are unable to improve the PCR specificity with the concentrations of 3 orders of magnitude more than that of PEI. The result shows that the surface charge polarity of PEI and PEI derivatives plays a major role in their effectiveness to improve the PCR specificity and efficiency. We also used PEI modified with both PEG and acetamide which could prevent additive from reacting with protein. We show that the PCR enhancing effect is primarily based on the interaction between the PCR components and PEI derivatives, where electrostatic interaction should play a major role to locally concentrate the PCR components on the backbones of the branched PEI, enabling enhanced PCR specificity.We also studied the effect of molecular morphology on PCR reaction. PEI is highly branched polymer with soft and flexible characteristics. Our laboratory synthesized the PEI-entrapped gold nanoparticle(PEI-Au) with decreased PEI flexibility and a close-to-spherical shape. The improving effect of PEI-Au in the two systems is discussed.It is important to know how well the amplification fidelity is kept in PEI-enhanced PCR. We sequenced PCR products and compared their sequencing results with a tradition PCR. The results show that PEI-assisted PCR has the similar fidelity with traditional PCR.