Research On The Influence Of PCR Enhancer To Long DNA Assembly Efficiency

In the post-genome era, there are more and more studies that based on wholesequences. An accurate and fast approach to get long DNA samples is needed forscientists. So far, DNA assembly is the key technology for genome or long DNAsynthesis in vitro. But most of assembly methods have the shortcomings of high costand poor efficiency, and their use is limited by the length of the recombinationfragments. Overlap extension PCR(OE-PCR) and Gibson Assembly are both the typicalDNA assembly methods in vitro, and they have the common shortcomgs of mostassembly methods. In recent years, nano-materials and nuclear chemicals are playing amost important role in the amplification of DNA fragments, and both of them canimprove the efficiency of PCR by interacting with DNA template or DNA polymeraseand cutting down the Tm values. Inspired by these researches, we added nanomaterialsand nuclear chemicals into the reaction system of OE-PCR and Gibson assembly, andselected several of enhancers for the two methods.This subject includes four parts:(1) the obtaining of DNA fragments with overlaps,(2)basic assembly tests,(3)selections of enhancers,(4)preliminary study for theenhancing mechanism.DNA fragments with overlaps are obtained by PCR with overlap-primers. In theseparts,15pairs of overlap-primer were synthesized. The overlaps have three lengths of40bp,80bp and200bp, respectively. The length of recombination fragments ranges from6kb to27kb.Many studies have been done in the phase of influence test and selection of PCRenhancers. First, reaction concentration and conditions were tested, and then twoexperimental models were established. Second, the influence of different nanomaterialand nuclear chemicals were tested. Eventually,3kinds of nuclear chemicals and4kindsof nanomaterial were selected as the enhancer of Gibson assembly; one nuclearchemical was selected as a enhancer of OE-PCR.In the last stage, Q-PCR is adopted to analysis the enhancing mechanism of theseenhancers. The result showed that these enhancers can improve the specificity of OE-PCR, and cut down the Tm values obviously.These DNA assembly enhancers selected in this subject were all found for the firsttime. At the same time, this research laid a foundation for the future application of PCRenhancer in DNA assembly in vitro.