Amplified Detection Of Nucleic Acids Based On DNA Aligner-Mediated Cleavage

Nucleic acids are important biomarkers for molecular biology research.Therefore,the highly sensitive and specific detection of nucleic acids is essential in many areas,such as clinical diagnostics,forensic analysis and food safety.Currently,there already exist many methods for nucleic acids detection including polymerase chain reaction(PCR)and isothermal amplification.The traditional PCR requires expensive thermal cyclers,which largely limit its application in point-of-care testing.While isothermal amplification can work at constant temperature,most of them involve complicated design of primer and/or multiple proteins/enzymes.Thus,it remains necessary to develop more simple and rapid techniques for detection of nucleic acids.In this paper,two signal amplification strategies for rapid and sensitive detection of nucleic acids were developed based on DNA aligner-mediated cleavage,and one of the methods was preliminarily demonstrated for detection of uracil-DNA glycosylase activity.The main contents include:Chapter 1:This chapter reviews several methods of nucleic acids amplification,cascade signal amplification and nicking enzyme signal amplification for DNA detection.Chapter 2:Dual enzyme cleavage-based cascade signal amplification for nucleic acids detection was developed.In this system,a modified DNA aligner that contains an abasic site was first cleaved by Tth Endonuclease IV in the presence of target DNA,and then served as an aligner to mediate the cleavage of molecular beacon by nicking endonuclease Nt.BstNBI.These cascade reactions not only overcame the sequence dependence of Nt.BstNBI,but also improved the detection sensitivity.The results showed a good linear correlation between the fluorescence intensity and the logarithm of target DNA concentration ranging from 1 pmol/L to 1 nmol/L,with the detection limit at 0.52 pmol/L.Moreover,the proposed method also showed a good capability of identifying single base mutation in target DNA.In addition,this method also features simple probe design and excellent universality.By modifying a small fragment on MDA’s loop,it can be used to detect various target DNA.Experiments with target DNA spiked in human serum showed the potential of applying this method to real samples.Chapter 3:A single nicking endonuclease-based multiple signal amplification was developed for simple and rapid quantification of target DNA.The proposed method showed a linear detection range from 1 pmol/L to 1 nmol/L,with the detection limit at 0.76 pmol/L within 30 minutes and a good capability of identifying single base mutation in target DNA.In addition,this reaction system has the advantages of simple operation and low cost because only one enzyme is needed in the whole amplification process.Experiments with target DNA spiked in human serum showed the potential of applying this method to real samples.Chapter 4:The single nicking endonuclease-based multiple signal amplification was used for the sensitive detection of UDG activity.The results showed a good linear correlation between the fluorescence intensity and the logarithm of UDG concentration ranging from 0.01 U/mL to 1 U/mL.Chapter 5:The main achievements,certain shortcomings and expectation of this thesis are summarized.