Research And Optimization Of The Helicase-dependent Isothermal DNA Amplification

Helicase-dependent Amplification(HDA)is a new type of in vitro DNA amplification technology that uses the helicase to open DNA double-strands to achieve replication of the target fragment.It has the advantages of not requiring complex temperature control equipment at constant temperature and so on.In recent years,Helicase-dependent amplification has been applied to biomedical engineering such as rapid nucleic acid detection.However,compared with the traditional nucleic acid amplification technology,the activity and specificity of the helicase in the helicase-dependent amplification need to be improved,which leads to the problem that the target DNA amplification efficiency is not high.In order to improve the efficiency of DNA amplification,this study increased the substrate recognition activity of DNA helicase by designing the sequence of the primer end.In addition,the application of different types of DNA helicases and related proteins in the helicase-dependent amplification was also explored.The biochemical activity of helicase has shown that the DNA substrate end sequence affects the efficiency of the initiation of the double-stranded unwinding reaction,which in turn affects the amplification efficiency of the DNA product.In the process of DNA amplification,the primers anneal to the template strand and are polymerized by the polymerase to form a new DNA strand.The newly synthesized DNA strand is then used as a substrate for the reaction to enter the next cycle of amplification.Therefore,the effects of the enzyme can be improved by primer design.By designing primers containing different modifications at the 5' end and comparing their effects on the helicase-dependent amplification,the preference for the specific substrate end of the helicase-dependent amplification was found.With optimal primer modification,the isothermal amplification yield can be increased by 5-fold.In addition,this article also explored the helicase-dependent amplification based on different types of helicase and related proteases.Here,we performed molecular cloning and recombination of 11 typs proteases including UvrD helicase of Gram-negative Escherichia coli,Nucleic acid PcrA helicase of Gram-positive bacterium,and relatedly-active nucleic acid plasmid-replicating proteins,RepC and MutL.Protein expression was highly purified using a universal,specific peptide tag,and a helicase-dependent amplification reaction system based on different enzymes was constructed.The related biochemical activities were systematically analyzed,and the replacement of different helicase was explored.In summary,this article explores and optimizes the helicase-dependent amplification from multiple perspectives and finds that the amplification efficiency of the target product can be effectively improved by designing the amplification of the primer sequence.It can be expected that the results of this study will help to optimize the design of helicase-dependent amplification to a certain extent and advance its application in related biomedical engineering.