Research On Novel Isothermal Exponential Amplification Methods Based On Aligner-mediated Cleavage

The process study of cancer elucidates the mechanisms underlying the occurrence,development and evolution of cancer and plays a guiding role in the diagnosis,intervention and therapy of cancer.And the detection of the biomarkers of cancer is one of the most efficienct ways in this field.Nucleic acids are vital biomarkers closely related to many cancers,thus it is of great significance for effective screening and highly sensitive detection of nucleic acids in the process study and control of cancer.Consequently,the amplification and detection of trace nucleic acids in complex samples have attracted increasing attentions these years,especially the isothermal amplification techniques.Since early 1990s,various isothermal amplification methods have been developed and widely used for biosensing many targets such as DNA,RNA,proteins and so on.Among others,isothermal amplification methods based on nicking endonucleases(NEases),e.g.,strand displacement amplification(SDA),exponentialisothermal amplification reaction(EXPAR),nicking endonuclease signal amplification(NESA)and so on,have been intensively studied because of their simple mechanism,fast reaction rate and high amplification efficiency.However,the requirement for the specific recognition sequences of NEases in the target DNA leads to restricted versatility for these methods.What’s more,the notorious non-specific amplification that heavily influences the amplification performance is also an intricate problem.In this thesis,we first developed a novel aligner-mediated cleavage(AMC)technique based on a hairpin-shaped DNA-aligner(DA),which fully overcame the sequence limitation of NEases.On this basis,two novel isothermal amplification methods have been proposed,and the inhibition of non-specific amplification has been studied as well,which greatly improves the sensitivity and reproducibility of the proposed methods.At last,a novel steric effect-regulated isothermal exponential amplification was developed for fast and homogeneously sensing of proteins.The main contents are listed as follows:In chapter 1,the current isothermal amplification methods are reviewed.In particular,the NEases-based methods including their mechanisms,applications and limitations are presented in detail.In the end,the contents of this thesis are briefly summarized.In chapter 2,a novel strategy for programmable sequence-specific cleavage of nucleic acids using DA and NEases(Aligner-mediated cleavage,AMC)was described,DA consists of a stem-loop structure and two side arms,in which the specificrecognition site of a certain NEase is placed in the stem.Thus,it enables the loading of the NEase on DA’s stem,localization to a specific locus through hybridization of DA’s side arms with target DNA,and cleavage thereof.By using just one NEase,AMC can easily make a break at any specific locus and tune the cleavage site at single-nucleotide scale,fully overcoming the sequence limitation of NEases.In chapter 3,an AMC-based strand displacement amplification method(AMC-SDA)was successfully de-veloped.Thanks to the versatility of AMC,the proposed AMC-SDA also features excellent universality.Moreover,the initiation manner of AMC-SDA facilitates the adoption of 3’-terminated primers,which restrains the non-specific amplification effect to some extent,thus endowing AMC-SDA with high sensitivity,high specificity and simple primer design.The good performance.of AMC-SDA in detecting plasmid and HBV DNA in clinical samples indicates a promising application potential.In chapter 4,the factors influencing the performance of AMC-SDA were first discussed,and the optimization strategy was proposed accordingly.By optimizing the structure of DA,adopting phosphorothioated primers and introducing abasic sites into primers,AMC-SDA was able to detect as low as 10-17 M target sequence with high reproducibility.This is ascribed to the elimination of DA’s/primer’s extension along the target sequence,the suppression of the degradation and the non-specific amplification effect of DA/primers.In chapter 5,in order to address the sequence limitation of EXPAR,it was coupled with AMC to form a new method called "aligner-mediated cleavage-triggered exponential amplification"(AMCEA).Compared to original EXPAR,the universality of AMCEA is greatly improved,and can be used in amplified detection of various nucleic acids.Results show that AMCEA can sensitively detect amol-level target sequence in serum,indicating a good application potential.In chapter 6,by combining the DA-regulated the amplification with the steric-effect,a novel isothermal exponential amplification method(steric effect-regulated EXPAR,SER-EXPAR)was proposed for one-step,fast and homogeneously sensing of proteins.SER-EXPAR can sensitively and selectively detect nanomole target proteins in serum,indicating a certain application potential.In chapter 7,the innovation as well as some future expectations of this thesis were summarized.