Nucleic Acid Detection Based On Enzymatic Reaction And Signal Amplification

Nucleic acid detection covers genetic screening, disease diagnosis, biomedical research and other fields, and is closely related to social stability and human health. How to overcome the disadvantages of the traditional nucleic acid detection methods is very important, such as, complex operation, time-consuming, low sensitivity defects. Meanwhile, to pursue high sensitivity, high precision, quick and easy method is a hot issue for many researchers. In order to meet the urgent demand of highly sensitive detection of DNA, signal amplification detection technology arises at the historic moment, which combines various enzymes as tools and with the help of some special materials such as nano-particles, or combined with electrochemical and optical technology to achieve highly sensitive amplification testing. In this paper, three different signal amplification strategies have been developed based on hybridization of nucleic acids and the signal amplification technology.1. Signal amplified detection of DNA based on the DNA-HRP conjugation and gold nanoparticlesWe developed a HRP assisted signal amplified assay for DNA detection based on sandwich hybridization, which employed HRP-catalyzed decomposition of hydrogen peroxide ability to output a detectable signal through the aggregation and dispersion of gold nanoparticles. In presence of target DNA, firstly, the target strand hybrid with HRP-labeled secondary chain, then they fixed to the biotinylated streptavidin agarose beads, and the HRP decomposed hydrogen peroxide, finally, gold nanoparticle aggregates to output detectable signal. This method used HRP catalytic reaction for signal amplification, and realized the signal amplifid detection of nucleic acid. The limit of detection is10nmol/L.2. Label-free fluorescence amplifying detection of DNA based on hairpin probe and Exo IIIA novel label-free fluorescence signal amplifying method for DNA detection was developed with high specificity and sensitivity based on a long arm hairpin nucleic acid probe and exonuclease III. Without the target DNA, the SYBR Green I dye could be embedded into the stem of hairpin nucleic acid probe to generate strong fluorescence. While in the presence of target DNA, exonuclease III could catalyze the stepwise removal of mononucleotides from3’-OH termini of double-stranded DNA with the degradation of the hairpin probe. Then the SYBR Green I was continuously released, resulting in fluorescence intensity decreased. It realized the label-free signal amplifying detection of DNA. The detection limit of this method was as low as320fmol/L. It can be expected to provide a novel, simple, label-free and rapid tool for DNA detection.3. Exonuclease Ⅲ assisted target recycling amplification assay for high specific detection of SNPWe developed an amplification and simple method for detection of SNP. In this method, a nucleic acid probe labeled with FAM fluorophore was designed, and combined with exonuclease Ⅲ specific cleavage of3’-OH termini of the double-strands. When in presence of target DNA, the signal probe is perfectly complementary to the target DNA, and then the duplex hybrid induced the exonuclease Ⅲ to degradate the signal probe and the targets released for the next cycle. While in absence of target DNA, it cannot induce the degradation of exonuclease Ⅲ, which lead to fluorescent quenching. This method showed excellent detection abilities, the detection limit was as low as137pmol/L...