Characterization And Application Of Taq DNA Polymerase Fused With A CL7 Protein

Taq DNA polymerase,as a very valuable tool enzyme in biotechnology,plays a vital role in PCR technology.The emergence of thermostable Taq DNA polymerase have overcome the limitation of its resistance to high temperature,thus having promoted the rapid development of PCR technology,which is not only widely applied in medical fields,including early diagnosis and prevention of cancer,detection and identification of pathogen,prenatal diagnosis and forensic identification,but also accelerated research related in biological fields,such as genotyping,molecular cloning,site-directed mutagenesis,DNA sequencing and DNA polymorphism.With the increasing replacement of biotechnology,existing Taq DNA polymerases cannot meet the needs of modern in vitro diagnostics,researchers have proposed many methods to improve enzymatic properties.Here,we fused CL7 protein with Taq DNA polymerase by genetic engineering and express the recombination protein in this study.CL7 is the mutant of CE7 nuclease domain from E.coli,remaining the ability to binding DNA.In this project,the recombination CL7-Taq DNA polymerase performed better enzymatic properties,including thermostability,extension rate,the sensitivity of template and the resistance to inhibitors.CL7-Taq DNA polymerase also showed higher sensitivity in the research fields of mouse genotyping and cancer-related gene detection.And our current results fully demonstrate that CL7-Taq DNA polymerase can quickly and accurately detect mouse genotypes without extracting genomic DNA.CL7-Taq DNA polymerase can not only amplify by single copy DNA template containing inhibitors,but also significantly improve the resistance to blood samples.Therefore,CL7-Taq DNA polymerase has broad application prospects in the molecular biology and medical diagnostic field including medical diagnosis and infectious disease detection.And our study can provide a new research method for enzyme engineering of Taq DNApolymerase.The main contents of this study are as follows:1.Expression and purification of CL7-Taq DNA polymeraseFirstly synthetic CL7-Taq gene was cloned into the p ET-30 a vector to obtain the recombined plasmid p ET30-CL7-Taq to express CL7-Taq protein.Then it was transformed into E.coli BL21(DE3),single strain was selected and induced to express recombinant protein.CL7-Taq DNA polymerase was purified by Ni-affinity chromatography.2.Characterization of CL7-Taq DNA polymeraseThe enzymatic properties of CL7-Taq DNA polymerase,including thermostability,amplification rate,template sensitivity,and tolerance to PCR inhibitors,were compared with wild-type Taq DNA polymerase respectively.The results showed that the thermostability and the resistance of inhibitors of CL7-Taq DNA polymerase were significantly enhanced.The PCR amplification rate is increased up to 4 kb/min,which is twice that of wild-type Taq DNA polymerase,and the template sensitivity is also increased by about one hundred times.3.The application of CL7-Taq DNA polymeraseIn order to explore the application of CL7-Taq DNA polymerase in clinical diagnosis,this study tried to detect single nucleotide mutation(SNPrs12516)of breast cancer gene(BRCA1)with Amplification Refractory Mutation System(ARMS-PCR).The sensitivity of template detection and blood samples resistance of CL7-Taq DNA polymerase has increased remarkably.PCR can be performed directly using a blood sample as a template,without the pretreatment of sample,which not only reduces the cost but also greatly shortens the detection time.In addition,we also used CL7-Taq DNA polymerase for genotyping studies and successfully detected mouse genotypes using single copy DNA template.