Research Of Pfu DNA Polymerase Improvement For Real-Time Fluorescent Quantitative PCR

Since SaiKi et al.used Taq thermostable polymerase in PCR reaction in 1988,this polymerase gave a big boost to the PCR technology development,and promoted the rise of Real-Time Quantitative PCR technology.Molecular biology developed dramatically.With the popularization and application of PCR technology,people gradually realized the disadvantages of Taq polymerase,its low fidelity is not suitable for high fidelity PCR and low amplification ability of complex sample requires sample treatment and PCR system optimization and so on.B family Pfu polymerase has high fidelity,high processivity and strong resistance to interference.It doesn't has 5 'to 3'exonuclease activity but has strong 3 'to 5' exonuclease activity which may hydrolyze free probe and influence fluorescence signal detection,and that leads Pfu polymerase not suitable for qPCR.So the aim of this study is improving Pfu polymerase for qPCR detection of complex samples.Based on the structure and properties of Pfu polymerase,we adopted two kinds of improvement strategy.First,we performed site-directed mutagenesis with Pfu polymerase and obtained Pfu-mut polymerase with weak 3 'to 5' exonuclease activity and couldn't hydrolyze free probe to effect the fluorescence signal.And we screened molecular beacon probe-which does not based on the activity of exonuclease activity and hydrolysis-for qPCR system.Second,we fused Taq polymerase N-terminal 5 'to 3'exonuclease activity domain to the N-terminal of Pfu polymerase,and obtained the TNF-Pfu-mut polymerase with 5 'to 3' exonuclease activity,which can be used for ordinary Taqman probe qPCR system based on hydrolysis.In addition,we also screened Pfu polymerase antibody,complex with antibody can help the hot-start effection and statement of Pfu polymerase.We found that the improved Pfu-mut polymerase have higher sensitivity for serum samples in qPCR detection than HS-Taq polymerase of Takara about one order of magnitude(101 fold),and it can tolerate at least 75%(vol/vol)extracted serum sample without affecting qPCR sensitivity.The amplification results of high GC content targets showed that Pfu-mut amplification sensitivity is obviously better than HS-Taq about three order of magnitude(103 fold).TNF-Pfu-mut manifests that exonuclease and polymerase activity can work properly.But its sensitivity for serum samples in qPCR detection is lower than HS-Taq polymerase.High GC content targets amplification result is not good enough,and it needs more improvement.To sum up,this study improved the Pfu polymerase and applied it to the qPCR detection,verified the advantages of its performance in complex samples qPCR.And it helped to improve the future study and application of B family polymerase and provided more selections for qPCR polymerase.