| Directed evolution is a process in which human beings imitate the natural evolution and carry out protein modification in the laboratory by means of molecular biology so as to realize the rapid evolution of biological proteins.As the first step,nucleic acid molecular mutation has played a vital role in the directed evolution of proteins,it determines the diversity function of the modified protein.Among numerous DNA molecular mutagenesis,the use of circular single-stranded DNA(C-ss DNA)to construct of a mutant protein is a promising mutation technology.C-ss DNA is able to introduce the mutation point by highly efficient annealing with mutant oligonucleotides.Since it is directly operated on plasmid molecules,molecular cloning steps are generally not required in the later operation process.The early preparation methods of C-ss DNA mostly relied on phage assisted production,which faced many problems such as cumbersome experimental process and complex preparation process.In order to facilitate and efficiently construct protein mutants,homing endonucerase I-Hmu I was used in this study to efficiently prepare C-ss DNA,so as to develop a more convenient nucleic acid mutation system.In addition,in the optimization process of nucleic acid mutation system,the heterologous double-chain products were amplified by using the method of multi-cycle amplification to improve the low mutation rate in the mutation scheme.The results showed that lab-purified nickase of I-Hmu I and exonuclease of Exo III could efficiently prepare C-ss DNA.At the same time,with the increase of the number of amplification rounds of heterologous double-stranded molecules,the efficiency of nucleic acid mutation system was significantly improved,reaching about 70% of the mutation efficiency.Therefore,this study proposes a feasible mutation scheme,which can rapidly realize the construction of mutants,and provides a new idea for the related research of protein and nucleic acid molecules. |
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