Enhancing Polymerase Chain Reaction Efficiency by Nuclear Small Chemicals
|School||Harbin Institute of Technology|
|Keywords||PCR additives nuclear chemicals nucleotide amino acid|
With the development of PCR technology, more and more scientists had diverted their attentions to the new finding of PCR enhancers. In this subject, nearly 100 hundred nuclear chemicals had been selected from the pathway in nuclear of Saccharomyces cerevisiae by the method of bioinformatics. Most of the nuclear chemicals were amino acid, nucleotide and their analogues. After large-scale tentative experiments, 7 kinds of nuclear chemicals had been selected for their significant amplification. Furthermore, series of tests had been done for the mechanism research of these nuclear chemicals. The results demonstrated that these nuclear chemicals are likely to become the new PCR enhancers and promote the development of PCR technology.This subject include four parts: search and select relative nuclear chemicals, experimental tests, preliminary study for the enhancing mechanism and test for the sensitivity and stability of the PCR system.For the searching part, 96 kinds of nuclear chemicals had been selected form the nucleus pathway of Saccharomyces cerevisiae and a mount of tests had been done with two experimental models. Eventually, 7 kinds of nuclear chemicals among them had been selected.In the following study, real-time Q-PCR and DNA replication fidelity test were adopted to analysis the amplification mechanism of these 7 kinds of nuclear chemicals. The results demonstrated that most of the nuclear chemicals can promote the replication rate of DNA and cut down the Tm values obviously. For the fidelity test, These 7 kinds of nuclear chemicals didn’t make great impact for the DNA replication.Finally, series of tests had been done to detect the sensitivity and stability of the PCR system. The result showed that the sensitivity of these nuclear chemicals were nearly 10 times higher than the common PCR system and this system can also make the repeated PCR continued to the third round.The enhancing effect of these 7 kinds of nuclear chemicals were all the first time finding by our laboratory and had been protected by the patent. We hope the nuclear chemicals can be made to the PCR kits and contribute to much more laboratories.