SNPs Assay Based On Nucleic Acid Enzyme Signal Amplification

Nucleic acid probes have been increasingly applied in a broad range of fields in chemistry, biomedicine, disease diagnosis and treatment due to their simple design, good stability, easy synthesis, and flexible signal mechanism. In recent years, signal amplification, especially ribozyme combined with other analysis technology of signal amplification technique, due to its simple, sensitivity and specificity, and relatively short response time, have been developed rapidly in the analysis of trace substances, such as nucleic acid, protein, and heavy metals. In this thesis, novel and sensitive detection methods had been designed combining nucleic acid enzyme signal amplification and flexible signal mechanism of nucleic acid probes. The contents of the thesis are as followed:(1) A novel double amplification detection of single-nucleotide polymorphisms had been developed based on cleavage reaction and singlet oxygen. In the presence of the perfect-matched target DNA, the probe was hybridized with it and released target DNA duo to RNase H cleavage reaction. Then the released target DNA could hybridize to another beacon. After many cleavage cycles, the probes were absorbed single-walled carbon nanotubes surface and had low photosensitive activity. However, if the mismatched target was present, probes were not absorbed single-walled carbon nanotubes surface and Ce6 could continual produce singlet oxygen. This assay implemented highly sensitive, and specific detection of single-nucleotide polymorphisms by fluorescence intensity difference in solution.(2) We have developed a novel label-free and sensitive detection method for single-nucleotide polymorphisms assay using G-quadruplex-hemin DNAzymes and RNase cleavage reaction. In the presence of the perfect-matched target DNA, the probe was hybridized with it and released target DNA duo to RNase H cleavage reaction. the released target DNA could hybridize to another beacon. After many cleavage cycles, the two G-quadruplex halves of the cleaved beacon dissociated from the target and di?used away from each other, resulting in the very low peroxidase-mimicking catalytic activity. However, if the mismatched target was present, high peroxidase-mimicking catalytic activity was maintained after the cleavage reaction step. This assay method implemented label-free, colorimetric, and amplification detection of single-nucleotide polymorphisms by absorption intensity difference in solution.