Combination Of Nucleic Acid Probes And Nucleases For Analysis

Nucleic acid probes are ideal molecular, engineering tools for a variety of applications in biology, chemistry and biomedicine due to their easy synthesis, good stability, simple design and flexible signal mechanism. The combination of nucleic acid probes and nucleases can improve the detection sensitivity, specificity, and this strategy can be also used to monitor the interaction process of biomolecules in real time. The thesis has developed some novel detection methods based on nucleic acid probes and nucleases. The contents of the thesis are as followed:1. A novel amplified method for small targets detection based on double-stranded fluorescent probe and exonucleaseⅠwas developed. Double-stranded fluorescent probes, formed by 3'-TAMRA-aptamer and 5'-DABCYL-cDNA, had low fluorescent intensity as ExonucleaseⅠonly cut ssDNA from 3'to 5'specifically. When the target was added, the Double-stranded probe was cut by exonucleaseⅠonce the aptamer-target complex formed and the fluorescent intensity increased. The released target bound to another aptamer and started another cycle. By monitoring the fluorescent intensity change, adenosine was detected with high specificity and sensitivity. The limit of detection is 0.65μM. This method has a potential application in other small targets detection.2. A new method for detection of APE1 based on molecular beacon probes with AP site was developed. Two types of molecular beacon probes were designed. P1 was a normal molecular beacon probe with an AP site at loop and P2 was a long-stem molecular beacon probe with a AP site at stem. P1 and P2's performance to detect the APE1 activity was compared and the superior p1 probe was used to detect the APE1 activity. The linear-detection range is 0.25-50 U/mL, and the limit of detection is 0.25 U/mL. This assay can detect APE1 rapidly and sensitively.3. A new method for zinc detection based on molecular beacon and S1 nuclease was developed. S1 nuclease cut single strand nucleic acids specifically. This method took advantage of the dependence of S1 nuclease activity on zinc concentration. Molecular beacon was employed as the substrate. By monitoring the initial velocity change of S1 nuclease with different zinc concentrations, zinc detection could be achieved. The limit of detection is 120μM. The method is,quick and simple since no complicated ion carrier synthesis was needed.