Application And Research Of Novel DNA Molecular Machine

In the field of biochemical analysis research, the development of new signal amplification technology has become an important research direction for detection of target molecules that can’t be detected because of its low concentration. The signal amplification technology includes biological barcode signal amplification technology, ribozyme combined with other analysis technology of signal amplifier technique and so on. In this paper we combined fluorescence techniques with DNA molecular machines, nucleic acid aptamer recognition technology, target substitution circle amplification method based on DNA enzymes identify components and enzyme circle amplification method. And we realized high selectivity and sensitivity for the detection of bioactive small molecules and target DNA. The paper includes the following chapters:1. We built multiple DNA molecular machines based on isothermal circular strand-displacement polymerization for the fluorescence detection of adenosine triphosphate. First, aptamer combined with adenosine triphosphate to release primers. Then, under the action of Klenow polymerase, primers extended to release fluorescent probes and formed double-stranded DNA. The restriction enzyme specificity cut two recognition sites on the double-stranded DNA, and further caused strand-displacement reaction to release more fluorescent probes. Under the optimum reaction conditions, the detection limit of the experiment is 1.O×10-8 mol-L-1. So the experimental method can be used for the detection of ATP in actual samples. The experimental method is simple and has high selectivity and sensitivity.2. Based on rolling circle amplification and catalytic hairpin assembly, we designed a new experimental method for ultrasensitive fluorescence detection of target DNA. First, target DNA and padlock DNA hybridized. Under the action of T4 DNA ligase, circular DNA was formed. Then, under the action of Phi 29 DNA polymerase, product of rolling circle amplification reaction contained hundreds of tandem-repeat catalytic sequences for the hairpin assembly. The reaction released a lot of fluorescent probes with two-step circle amplification technology and achieved the sensitive detection of target DNA.3. Based on the rolling circle amplification, we designed DNA molecular machines to realize the sensitive detection of target DNA. First, target DNA and circular DNA hybridized. Then, under the action of Phi 29 DNA polymerase and dNTPs, a lot of DNA was produced. Then the new DNA and hairpin probe hybridized, and stem ring structure of hairpin probe was broken and further combined with circular DNA. Rolling circle amplification reaction produced large amounts of repetitive DNA fragments which combined with the signal probe. When there was Mg2+ in the reaction system, Mg2+ can specificity cut recognition sites on the chain and increase the distance between the fluorescence quenching and reactive. We realized the fluorescence signal detection of target DNA.