Site-directed Mutagenesis Of Thermostable DNA Polymerase And Bio-panning Of Nanobody Applied To Hot-start PCR

DNA polymerase was originally discovered by basic molecular biology research on the molecular mechanisms of DN A replication in living organisms. As we all known, DNA polymerases synthesize new DN A strands according to the template DNA, using deoxynucleotide triphosphates during DN A replication. DNA polymerase catalyzes phosphodiester bond formation between the terminal 3’-OH of the primer and the a-phosphate of the incoming triphosphate. C urrently, many thermostable DNA polymerases are used for PCR and other procedures that involve the copying of nucleic acids. These include multiplexed PCR, nested PCR, reverse transcription PCR, and DN A sequencing among others. Polymerases are also used to incorporate the modified nucleotides, including those labels, reports, or signal the presence of product DNA. These allow nucleic acids to be amplified from complex samples, including blood, saliva, forensic traces, a nd fossil remains. The choice of a particular polymerase depends on the specific need, especially for processivity and fidelity, temperature of initiation, or ability to accept unnatural nucleotide analogs. Each polymerase has different features, resulting from origin and genetic modification. However, the rational choice of the adequate polymerase depends on the application itself. The first part in this study, high fidelity DNA polymerase was modified using overlap extension PCR- mediated site-directed mutagenesis techniques by genetic engineering method, and was overexpressed in E. coli. The aim of this study is to enhance the ability of incorporating nucleotides to modified substrates(eg deoxynucleotides, dideoxynucleotides, ribonucleotides, and acyclonucleotide). Hot-start PCR can be achieved by using Taq DNA polymerase monoclonal antibody. It results to reduce primer mismatch, the formation of primer-dimers and the generation of non-specific amplification, increase the specificity of PCR. The second part of this experiment, with DN A polymerase as the target molecule, using bio-affinity panning method to obtain DNA polymerase nanobodies from alpaca natural phage libraries, and binding molecular cloning techniques, enzyme antibodies were expressed and purified, in order to apply to hot-start PC R, the main contents and results are as follows:1. By overlap extension PCR-mediated site-directed mutagenesis techniques, six mutations were introduced into the DNA polymerase gene sequence. Four recombinant plasmids were were successfully constructed, they are pET22b-polAD146AE148 A,p ET22b-polAD146AE148AA490 L,p ET22b-polAD146AE148A413LYP415 SAV and pET22b-polAD146AE148A413LYP415SAVA490 L respectively.2. We selected E. coli BL21(DE3) as an expression strain among three host strains E. coli BL21(DE3), E. coli Rosetta(DE3) plysS and E. coli BL21 CodonPlus(DE3) RIL. The optimal induced expression conditions are 0.1 mM IPTG induction at 37℃ 3 h. And the optimal PC R reaction buffer components of the recombinant DNA polymerase were as follows: p H 9.0, 5 mM(NH4)2SO4, 1.5 mM Mg2+. Meanwhile, recombinant DNA polymerase has a strong heat resistance, and recombinant DNA polymerase that treated for 6 h at 95℃ still keeps activity. Protein concentrations were determined by Bradford method, the gradient titration homemade enzyme activity was 7.5 U/μL, the specific activity was 18293 U/mg.3. Using Taq DNA polymerase as the target protein, five specific binding nanobody were screened by using the method of solid phase panning; using self- made DNA polymerase as the target protein, five specific binding nanobody were abtained by using the method of solid phase panning.4. By molecular cloning technology,prokaryotic expression vector of pET25-T6, pET25-T19 and p ET25-S3 were successfully constructed, and was transformed into E. coli Rosetta(DE3) plysS host strain, after the induction of 0.2 mM IPTG, the soluble protein of T19, T6 and S3 were obtained. Identified by ELISA, S3 protein bind to homemade enzyme with high affinity, but not binding in the enzyme active center, and therefore can not be used for hot-start PCR.