Preliminary Study And Application Of Nucleic Acid Isothermal Strand Displacement Amplification Technology Based On Bst Polymerase

As the carrier of genetic information and the encoder of protein,nucleic acid is one of the most basic composition of life,the occurrence of diseases and tumors is accompanied by changes in the level of nucleic acids in organisms.However,nucleic acid often exists in trace form.Therefore,the development of DNA amplification technology with high sensitivity and strong specificity is of great significance to biological analysis and biomedical research.The nucleic acid isothermal strand displacement amplification technology has attracted great interest in recent years by virtue of relatively simple strand migration principle,high efficiency and controllability on kinetics.With the development of molecular biology,Bst polymerase with strand displacement activity designed based on genetic have broaden horizons for strand displacement amplification technology.ttTherefore,the main research idea is to introduce Bst DNA polymerase into the enzyme-free nucleic acid isothermal strand displacement amplification technology,change the reaction model of traditional strand displacement amplification,convert the output type of nucleic acid signals,and improve the efficiency of amplification.The research work is as follows:(1)Developed a hybridization chain reaction mediated by Bst polymerase(pHCR).Compared with the linear growth of hybrid chain reaction(HCR),the exponential amplification mode of pHCR greatly improves the efficiency.The intervention of Bst polymerase changed the signal output type of the traditional hybrid chain reaction,transforming hundreds or even thousands bps of ds DNA into fixed-length short ds DNA,bringing new design space for signal application methods.A series of investigations and optimizations were employed on the pHCR,the detection limit of nucleic acid was down to pM level.(2)Developed a Bst polymerase mediated multiple self-folding strand displacement(pMTHSP)strategy.Based on the principle of multiple strand replacement amplification,it changes the signal output type and improves the amplification efficiency.It is proved that in the self-folding strand displacement amplification reaction,compared with dSpacer modification,the amplification efficiency induced by PS modification is higher,and pMTHSP can be applied to a wider range of temperature conditions than PS-THSP.Combining flow cytometry to quantitatively detect miRNA,the detection limit can reach pM level,and the simultaneous detection of two miRNAs is realized.The pMTHSP has good applicability for detection in actual serum samples,and the detection results are not significantly different from qRT-PCR.In summary,this article is based on nucleic acid isothermal strand displacement amplification technology,combined with high-efficiency amplification performance and strong strand displacement ability of Bst polymerase,and developed an isothermal strand displacement amplification technology mediated by Bst polymerase.Significantly improve the amplification efficiency,ameliorate the signal qualitative and quantitative capabilities,and bring new research ideas for nucleic acid amplification and detection.