| Modern science thought that the development of many diseases is directly correlated with the gene structure and expression changes. However, how to sensitively and specifically obtain these information in real time, especially obtain the change of gene expression products by utilizing effective tool, has become a great challenge for medical science and provide a new chance for the crosslink of analytical chemistry with medical science at the same time. The fluorescent molecular probe technology, a kind of sensitive and accurate research tool and method, which utilized the biochemical specificity, including nucleic acids hybridization, interaction of DNA-protein, specific recognization and digestion of enzyme to substrates et al, can transfer the information of bio-molecular into easily detectable fluorescent signal and is hopeful on the detection of gene expression products. However, the methods which can be applied to detect the gene expression are limited for the complex of products and lack of fluorescent probes. So, further develop new methods, new technology and new principles, which can be applied on detection of gene expression products, is a meaningful work of prospective research.The dissertation, which focused on the detection of gene expression products, was begun from the detection of uracil-DNA glycosylase (UDG) by using the enzymatic activity of these proteins and combing with the development of new kinds of probes. A series of fluorescent methods not only for detection of base damage repair protein including UDG, APE1, T4DNA ligase and hOGG1, but also for the quantitative detection of mRNA was developed. The simple, fast and real time new fluorescent probe technology for the detection of gene expression products, overcomes the drawbacks of traditional methods, which need the complicated step of isotope labeling, electrophoresis and autoradiography and is incapable of acquiring the dynamic data in real time. The main details are presented as following:Part 1 Detection of base damage repair protein based on fluorescent molecular probes1. A novel method for real time detection of UDG based on double-stranded fluorescent probesUDG is a kind of repair protein which is important for inhibition of gene mutation and immunological regulation by removing damage base of uracil. Here, we introduce a novel method for monitoring uracil removal in real-time. The double-stranded DNA probes containing uracil residues, which FRET can occur, are used to monitor this process in homogeneous solution. The method not only overcomes the drawbacks of discontinuity, time consuming and complication which inherited in traditional assays of radioisotope and electrophoresis methods, but also can be applied to determine the kinetic constants of UDG. The low limitation of UDG assay is 0.033U/mL, the KM and kcat are 0.11μM and 4s-1 separately. In addition, the method has been applied to explore enzymatic mechanism and the effect of metal ions, chemical drugs and concentration of substrates on UDG activity. Finally, UDG activities in tumor cells were quickly and accurately detected by applying this method in vitro. The work not only confirmes the feasibility of protein activity detection by fluorescent technology, but also provides a stable basement for making it as a common detection method.2. A novel method for real time detection of APE1 based on double-stranded fluorescent probesAs an important glycosylase of base damage repair, APE1 is associated with the processes of base repair, cell cycle regulation and cell apoptosis. In order to avoid the drawbacks of complication and time-consuming, which inherited in the traditional methods, a new method, which can detect APE1 activity rapidly, sensitively and precisely, was established by real-time monitoring hydrolysis of AP site in the homogenous solution. The linear-detection range is 0.024-2U/mL and the limit of detection is 0.024U/mL. Additionally, the enzymatic mechanism and kinetics were studied by investigating the influence of external factors including metal ions, chemical drugs and substrates on APE1 activity. The method was finally applied to detect APE1 activity of A549 and Tca8113 tumor cells in vitro.(3) Study the ligation mechanism of T4DNA ligase based on double-stranded fluorescent probes. DNA ligase is a vital enzyme in the repair, replication and recombination of nucleic acids. The ligation mechanism is usually assayed by electeophoresis and autoradiography, which is complex and time-consuming. Based on double-stranded probes, the ligation mechanism of T4 DNA ligase has been studied conveniently. The method can be applied to investigat the ligation mechanism of other DNA and RNA ligases.4. A novel simple and rapid method for real time detection of hOGG1 based on molecular beaconsEvidence showed that the difference of tumor susceptibility is often caused by the change of hOGG1 activity. A new method, which can rapidly, sensitively assay the hOGG1 activity, was established based on molecular beacons. The method has been applied not only for investigating the effect of external factors including metal ions, chemical drugs and substrate on hOGG1 activity but also for studying the enzymatic mechanism and kinetics. It was further applied to detect hOGG1 activity of MCF-7, A549, Tca8113 and HNE1 tumor cells. Comparing with methods of double-stranded probes, it avoided the step of preparing duplexes.Part 2. Detection of gene mRNA based on molecular beacons5. Quantitative detection of p33ING1 mRNA based on molecular beaconsMolecular beacons, which contain complimentary sequences with conserve region of ING1 mRNA, are applied to construct a standard curve of ING1MB/RNA after hybridizing with ING1 cRNA. With the help of standard curve, the effects of drug treatment, gene transfection and p53 RNAi on ING1 mRNA levels were quantitatively detected in homogenous solution after investigating the influence of metal ions, pH value and other nucleic acids on ING1 MB/mRNA hybridization. The results were further confirmed by RT-PCR. Compared with RT-PCR and other methods, the simple and time-saving method showed high accuracy and reproducibility by avoiding the amplification step.6. Quantitative detection of p21 mRNA based on molecular beacons (MBs)The p21 mRNA levels were detected based on molecular beacons in order to further broaden the application of molecular beacons detection technology. Molecular beacons, which can specifically hybridize with conserve sequences of p21 mRNA were designed and a p21MB/RNA hybridization standard curve was constructed according to the fluorescent change of p21MB caused by different concentrations of p21 cRNA. After investigating the influence of many factors including ions, pH and nucleic acids molecules on this method, the p21 mRNA levels of tumor cell with drug treatment, gene transfection and p53 RNAi were quantitatively detected with the help of standard curve. From the results, it can be found that the simple technology can directly detect target mRNA without further steps of transcription and amplification.7. Detection of ING1 mRNA and biological characteristic analysis of HNE1 cellsThe fluorescent changes of ING1 molecular beacons caused by mRNA of HNE1 cells with gene transfection or not were detected after treatment with 5-fluoruracil. The intensities of fluorescent change were used to stand for the ING1 mRNA levels. In addition, the effect of ING1 mRNA levels on cell biological characteristics was studied by combing with other analytical tools including cell growth detection, flow cytometry, western blotting and MTT assays. The results showed that ING1 mRNA levels can be acted as an indicator of cell growth speed, distribution of cell cycle, protein expression and cell sensitivity to 5-fluoruracil.
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