Isothermal Amplification Of Circular DNA Molecules Based On Phi29 DNA Polymerase And Cre Recombinase

Isothermal amplification is a process in which nucleic acid molecules were amplified at a constant temperature in vitro,compared with the traditional PCR amplification method,the requirement for equipment is low,low energy consumption,short reaction time,can meet the demand of rapid and simple,thus widely used in the field of clinical diagnosis,point-of-care(POC),whole genome amplification,single nucleotide polymorphism analysis,etc.Among the current major isothermal amplification methods,Rolling circle amplification(RCA)has attracted much attention because of its simplicity and rapid amplification.In this paper,we coupled RCA with site-specific recombination to study the amplification of circular DNA molecules in vitro,different from the traditional linear product,the product is circular DNA molecules,which have important significance to improve the existing isothermal amplification system.During the preparation of proteases used in the experiment,we explored the expression conditions of Phi29 DNA polymerase,homing endonuclease I-Hmu I and Cre recombinase,founding that the induction temperature and IPTG concentration significantly affects the expression conditions of protein,lower temperature and IPTG concentration will increase the soluble expression.Based on this,we finally confirmed the suitable expression conditions of 13 proteins such as Phi29,DNA polymerase,I-HmuI and Cre recombinase by a large number of experiments.According to the suitable expression conditions to extend culture,we have purified 12 proteins containing Phi29 DNA polymerase ultimately.In the purification process,based on small ubiquitin like modifier protein(SUMO)fusion tag technology,we further optimize the purification method of natural N terminal protein,and purified Phi29 DNA polymerase and its variants(T15I,N62D),Cre recombinase by the method.The concentration of the protein was determined by optical absorption,and the purity of the protein was analyzed by protein gel electrophoresis(SDS-PAGE).Subsequently,we analyzed the activities of Phi29,DNA polymerase,I-Hmu I and Cre recombinase respectively,and optimized the optimum reaction conditions.Then,we coupled the Phi29 DNA polymerase with I-HmuI to amplify plasmid DNA which was used as substrate in vitro,and the optimal reaction conditions in which Phi29 DNA polymerase can use the gap produced by I-Hmu I to initial isothermal amplification.Finally,based on the above results,we try to coupled single strand nicking reaction mediated by I-HmuI,RCA mediated by Phi29 DNA polymerase,site-specific recombination mediated by Cre recombinase to study the isothermal amplification technology from circular DNA template to circular DNA product in one step reaction.