Mechanistic Studies Of Dendrimer Derivatives And Nanocomposites On PCR Enhancing Effect

Polymerase Chain Reaction (PCR) is an in vitro DNA amplification technology. With the rapid development of PCR technology over the last20years, a series of relevant PCR technologies were designed to be widely applied in the field of genetics and medicine, PCR has become one of the most basic experimental techniques in the modern biology laboratory. However, the bottleneck problems, especially the aspect of non-specificity, hinder the further application of PCR. Thus it is urgent to find methods to solve the issues. Because of the unique physical and chemical properties, nanomaterials have been applied in the PCR by more and more researchers, which opening up a new thought for the improvement of PCR technology and widening the range of application of PCR.Based on the previous experimental work of our laboratory, we attempt to explore the dendrimer derivatives and nanocomposites as novel PCR additives. We have studied the effect of the surface charge, structure and antifouling properties of the nanomaterials on the specificity and efficiency of PCR. and discussed the potential mechanism.We established three PCR systems to test the role of the additives in PCR. One is a nonspecific system which used mouse tail genome as template; the other one is a two-round nonspecific system that used Pseudomonas aeruginosa genome (PA) as template; the last one is a three-round nonspecific system using λ DNA as template. We first employ the different surface modified (polyethylene glycol and/or acetyl groups) dendrimers entrapped/or non-entrapped with Au NPs as additives in the mouse tail genome nonspecific system and the PA nonspecific system. The results show that the both PCR systems can be optimized by the additives. The dendrimer entrapped with Au NPs seem to more effectively enhance the PCR specificity. In addition, the modification of antifouling PEG significantly lowered the optimization capability of the corresponding dendrimers, while this inhibition effect can be significantly compromised by the existence of Au NPs. The optimization concentration of G5.NHAc-mPEG is more than3orders of magnitude higher than that of{(Au0)300-G5.NHAc-mPEG}. In this condition that both materials are nearly neutral charged, the optimizing effect may be due to the Au NPs-induced thermal conductivity. Meanwhile, for the positive charged dendrimer derivatives and nanocomposites, the electrostatic interaction between the dendrimer and PCR components should play a main role in the optimization of PCR.Then we used the different surface modified (polyethylene glycol and/or acetyl groups) dendrimers entrapped/or not entrapped with Ag NPs as additives in the three-round amplification system to study their optimizing effect. We showed that all the additives can optimize the system. and the optimization ability improves with the increase of Ag NPs amount. With the same molar ratio between silver atom and dendrimer, the optimum concentrations of the positive charged dendrimer nanocomposities are lower than that of neutralized dendrimer nanocomposites. The present study suggests that the thermal conductivity of the Ag NPs and the electrostatic interaction between the dendrimer nanocomposites and PCR components may simultaneously play a role in the PCR optimization.It is worth mentioning that the optimal concentrations of the two materials in the three test system are very close, indicating that the developed dendrimer derivatives or nanocomposites may be efficient, stable PCR additives for enhancing different PCR systems.