Study Of DNA Fluorescent Probes Based On Collaborative Hybridization

Fluorescent probe is widely used in the detection of various nucleic acid targets,because of its simple operation,low cost and fast detection.However,the nucleic acids with a particular sequence are trace and usually needed to be amplified to facilitate detection.Signal amplification strategies are used to detect enzyme activity,nucleic acid and single-base mismatch by molecular amplification techniques,many of which are achieved by strand displacement reaction.The identification of base mismatch provides an important basis for the diagnosis and treatment of diseases.If we can develop a probe with specific recognition ability for a single base,it will provide some help for the diagnosis of diseases in the medical community.In this paper,we used a cooperative reaction to design a signal-amplified DNA strand displacement switch and construct DNA base mismatch recognition probe.The feasibility and reproducibility of the probe were verified by fluorescence and polyacrylamide gel electrophoresis.Specific studies are as follows:(1)Study of DNA strand displacement switch based on dual targets response: In this chapter,a DNA strand displacement switch based on dual targets response is designed by using the cooperative reaction.The principle of this strategy is that two target strands with low-concentration and different sequence hybridize with the two ends of the probe to produce a cooperative strand displacement reaction,which provides a new branch point domain for the next strand displacement reaction.We verified the well feasibility and reproducibility of this strategy by fluorescence and gel electrophoresis characterization.We can regulate toehold length of the different oligonucleotides,and its different cooperativity can also be used in the construction of other sensors or molecular devices.In addition,we systematically studied the double cycle signal amplification strategy and obtained the single signal cycle amplification strategy.This strategy has the characteristics of low concentration,low cost,rapidity and simple operation,so it has potential applications in disease surveillance and diagnosis and treatment.(2)Study of DNA base mismatch based on dual targets response: In this chapter,on the basis of cooperative hybridization,we designed a DNA base mismatch recognition probe which based on dual targets response.The principle is that the complementary target strand hybridize with the both toehold ends of the probe,it cantouch off the strand displacement reaction and replace the quenching strand.We used different experimental methods to characterize the probe performance in detail.The concentration ratio of a fluorescent probe and the DNA target strand were optimized by gel electrophoresis and fluorescence test.The optimal proportion of the probe was 1/1.5,and the concentration of DNA target strand was 40 n M.According to the identification of 9 kinds of target strands,we proved the complementary target strands with the best response.The results show that the probe has well specific recognition ability and provides a detection method for nucleic acid target analysis with high similarity.