Molecular Modification Of DNA Polymerase And Its Application In Probe QPCR

Extraction-free TaqMan probe qPCR technology has a wide application prospects in the field of clinical rapid diagnosis and rapid food detection with the advantages of time-saving,high efficiency and accuracy.TaqDNA polymerase,as the core component of the technology,not only needs to be able to tolerate inhibitors in different samples,but also needs to have the ability to hydrolyze TaqMan probe.However,DNA polymerases with both properties are scarce.Therefore,it is of great significance to modify TaqDNA polymerase and apply it to extraction-free TaqMan probe qPCR.In this paper,based on the mutant Sso7d-Taq(?289)(hereinafter referred to as STaq),a new mutant pFEN1-STaqwas obtained by further modification using genetic engineering technology.After the enzymatic properties of pFEN1-STaqwere studied,hot-start pFEN1-STaqwas further realized by chemical modification method,and then the hot-start pFEN1-STaqwas applied to extraction-free TaqMan probe qPCR.The main studies and conclusions are as follows:(1)Molecular modification of TaqDNA polymerase.The pfen1 gene was designed and synthesized,and the recombinant plasmid p ET-28a-pfen1-staq was successfully constructed for prokaryotic expression by homologous recombination,and the soluble expression of pFEN1-STaqrecombinant protein was realized in the prokaryotic system.The conditions of prokaryotic expression of pFEN1-STaqrecombinant protein were optimized as follows:induction temperature of 18?,induction time of 12 h,and final concentration of IPTG of 0.1 mmol/L.The recombinant protein pFEN1-STaqwas purified by nickel ion metal chelating affinity chromatography.A total of 5.4 mg pFEN1-STaqwas obtained by fermentation on 3 L medium.(2)pFEN1-STaqenzymatic properties study.The polymerase activity and 5'-3'exonuclease activity of pFEN1-STaqwere determined.The effects of different components in the buffer solution on the two activities of pFEN1-STaqwere studied.It was found that the polymerase activity of pFEN1-STaqwas stronger at pH value of 8.8 and low salt concentration.Moreover,the high concentration of MgSO₄ was beneficial to the 5'-3'exonuclease activity.pFEN1-STaqhad a great thermal stability,and the thermal stability of the polymerase activity was better than that of the 5'-3'exonuclease activity.The optimal elongation temperature of pFEN1-STaqwas 68?,and the elongation rate could reach 3,000-4,000 bp/s.pFEN1-STaqhad a strong tolerance to inhibitors,at least 1.0 U/m L heparin sodium,2.5?g/?L astragalus polysaccharide,5.0 ng/?L humic acid and 40%(v/v)whole blood.(3)Preparation and properties of hot-start pFEN1-STaq.The optimum conditions for the preparation of hot-start pFEN1-STaqby chemically modification were as follows:the mole ratio of citric anhydride to pFEN1-STaqwas 2500:1,the reaction temperature was 37?,the reaction time was 4 h,and the reaction pH value was 9.0.The heat shock conditions of the hot-start pFEN1-STaqwere optimized as follows:heat shock temperature was 95?,heat shock time was 10 min.After chemically modification,the amplification specificity and sensitivity were significantly improved,although the polymerase activity and 5'-3'exonuclease activity of pFEN1-STaqdecreased by about 80%and 85%.(4)Application of hot-start pFEN1-STaqin extraction-free TaqMan probe qPCR.A extraction-free TaqMan probe qPCR system was established based on hot-start pFEN1-STaq,and the constitute of the optimal 1×buffer were as follows:10 mmol/L Tris-HCl,20 mmol/L KCl,5 mmol/L(NH₄)₂SO₄,6 mmol/L MgSO₄,0.05%(v/v)Triton X-100,pH 8.3;The optimal probe concentration was 0.30?mol/L;The optimum amount of hot-start pFEN1-STaqwas 2.5U;The optimal annealing extension temperature is 60?.The established method can be tested with a maximum tolerance of 8%(v/v)whole blood,and can be used for qualitative and quantitative detection of samples within 8%(v/v)whole blood content.The method had good stability,high sensitivity and specificity,with a minimum detection limit of 10-100 copies/?L at 8%(v/v)whole blood content.