Nucleic Acid Probe Technique Based On Conformation Transformation

Nucleic acid probes as basic technique means for modern molecular biology,compared with common bioassay method, possess intrinsic advantage such as betterselectivity, high sensitivity, good stability, rapid speed for analysis and so on, whichhave opened up a series of new bioassay methods and been widely applied in fields ofchemistry, biology, bromatology, environmental monitoring and medical diagnosis etc.This paper designed four different nucleic acid probes based on the configurationconversion of nucleic acid, which combined with electrical or optical detection method.The detailed content is described as follows:β-indole acetic acid (IAA), a kind of plant hormones, plays a role as an inducer oran inhibitor at different concentrations. In chapter2, a functional nucleic acid wasdescribed for the electrochemical detection of β-indole acetic acid. Based on thetraditional antigen-antibody sandwich immunoassay model, elastic ssDNAself-assembled on gold surface and a terminal single IAA-labeled oligonucleotidesignaling probe was developed. Utilizing this assay platform, not only improves thedisadvantages such as depending on highly expensive special instruments orcomplicated sample preparations existed in conventional assays, but also developes asimple, low-cost, specific analytical method for DNA detection by combining withantigen-antibody competitive immunoreactions. The sensor described here presented aflexible behavior and can be immobilized in defined locations of different materialsinterfaces.Cocaine is not only serves as a medical anesthetic but also a very popular drugwhich display great toxicity on the central nervous. In the needs for its rapid detectionin law enforcement and clinical diagnostics, in chapter3, we report a fluore scenceprobe of autonomous aptamer-based machine by introducing a hairpin sequencelabeled with two fluorophores and a recognition site for endonuclease into the cocaineaptamer sequence. Due to high affinity for aptamer of the cocaine binding site drives toform a folding structure and triggers replication by polymerase and scission by nickingenzyme, producing an enhanced fluorescence signal. Adopt a system of combiningsignal probe with template probe which effectively reduces the―signal loss‖. Theresults demonstrated that the presented strategy was highly sensitive with the dynamicresponse range from8.0×10-7M to8.0×10-2M. As many human genetic diseases related to mutation of nucleic acid bases, theidentification and quantitative analysis of single nucleotide polymorphism (SNP) isimportant in the areas of genetics and medicine to elucidate the causes of variousdiseases. Based on signal-template integration system above and neighboringanalytical tool, in chapter4, we explore a―Y‖junction structure probe and achieve alow fluorescent background and high sensitivity method for DNA detection.Meanwhile, the probe displays a strong ability for single-base mutation discrimination.In the presence of a target, which together with the assistant probe, can hybridize withthe MB and open its hairpin structure to form the―Y‖junction structure probe. Underthe joint action of the polymerase and endonuclease, this technology providing a trueand efficient target-triggered enzymatic recycling amplification signal. Moreover,compared with the first-generation junction sensing system, our proposed sensingsystem also afforded a more sensitive response, and a detection limit of10pM wasobtained.G-quadruplex is unique secondary structure formed from the terminal overhandstrand of telomere DNA, which is essential for the course of the telomeric DNAelongation by telomerase catalysis. Therefore, G-quadruplex is a potential target fortumor chemotherapy and anticancer drug development. A quenching of intermolecularparallel G-quadruplex fluorescent probe sensing system is described in chapter5. Thesignal of fluorescence can be effectively quenched by the G-rich nucleic acid probe.The structure of G-quadruplex and the signal of fluorescence are under regulated byhybridizing complementary DNA to the G-tetrads. In addition, ideally response totarget DNA detection of the real sample is achieved with a detection limit of5nM. Thesignaling probe with high selectivity and sensitivity can be used to determine oth eranalytes including metal ions, proteins as well as nucleic acids in the ways ofend-terminal modification, and provided an interesting alternative in the developmentof excellent analysis techniques.