New Methods For Biochemical Analysis Based On Isothermal Amplification And DNAzyme

Isothermal amplification of nucleic acids is a simple process,which can achieve nucleic acid amplification at constant temperature.As precise temperature control is not required,it is a powerful complement to the traditional polymerase chain reaction?PCR?.Therefore,isothermal amplification has been widely used in pathogen and miRNA detection,single nucleotide polymorphisms,DNA methylation and other nucleic acid analysis.With the introduction of functional nucleic acid molecules such as aptamers and DNAzymes,the application of isothermal amplification techniques has been extended to the fields of protein,cell,functional small molecule and metal ion analysis.At present,a variety of isothermal amplification techniques have been established,among which rolling circle amplification?RCA?and nicking enzyme-assisted isothermal amplification are widely used in various types of biochemical analysis.As the design is simple and flexible,they are efficiently integrated with other signal amplification technologies and detection systems.This thesis focuses on the biochemical analysis based on isothermal amplification and functional nucleic acid molecules,including the following five parts.In chapter 1,the principles and applications of various commonly used isothermal amplification techniques were briefly introduced.Then,the functional nucleic acid molecules and signal readout methods combining with isothermal amplification in biochemical analysis were summarized.Finally,the applications of RCA and nicking enzyme-assisted isothermal amplification in various analyte detection and the problems in actual sample detection were introduced.Meanwhile,the objectives and main contents of this thesis were put forward.In chapter 2,the combination of nicking endonuclease assisted SDA and exponential RCA was demonstrated to develop a simple and targeted HPV genome detection approach.By making full use of multiple enzyme nicking sites existing in the HPV genomes and SDA,a specific single strand DNA?ssDNA?generated,and then triggered RCA.During the whole procedure,no primers were added into the system and denaturation was not required.As reagent and temperature are compatible,the whole progress could be operated in one pot.We chose HPV11,16,18,the most common subtypes of human papillomaviruses,as a model to perform the genotyping.HPV18 was detected in Hela cell lines by this approach.In chapter 3,we presented a simple,low cost and efficient method for miRNAs detection by making full use of the rapid magnetic separation and mild amplification reaction of RCA.A fishhook probe was designed and anchored on the magnetic beads as a recognition component to capture target miRNAs and simply isolate them from sample matrix.By simple magnetic separation,excess probes as well as other nucleic acids were removed,thus nonspecific amplification and background signal was minimized.Single base mismatch was distinguished and miR-21 in cell lysates of MCF-7 was detected without total RNA extraction.In chapter 4,we designed a circular probe containing the substrate strand of DNAzyme and developed a new mode of RCA based on the specific cleaving activity of Pb²⁺-dependent DNAzyme and the signal amplification technology of RCA.Then,a simple and low-cost homogenous fluorescence analysis assay was designed for Pb²⁺ detection.The substrate strand was biotin-labeled and the streptavidin-coated beads were used to separate the cleavage product from the initial substrate strand by simple magnetic separation,so as to convert the Pb²⁺concentration into the concentration of cleavage product.Due to the high concentration of the initial nucleic acid probe,the effective signal amplification could be achieved by the short-time RCA.As both Pb²⁺-assisted DNAzyme cleaving reaction and RCA were carried out in homogeneous solution,it was beneficial to improve the reaction efficiency and thus obtain high detection sensitivity.Moreover,the influence of common interfering ions and water matrix was evaluated,and good selectivity was achieved.As the matrix interference was small,the proposed assay could be used for the detection of actual samples.In chapter 5,we summarized the present work in this thesis and proposed the research content that need further improvement and subsequent research plan.