| Recently, novel fluorescent probe technology has already penetrated into many aspects of chemistry, medicine and biology and other fields based on its advantages of high sensitivity, specificity and simple operation. Several kinds of targeted molecular beacons in this paper have firstly designed and synthsised, then they were applied for quantitative detection and anlysis of target molecules including miRNA-21 and UDG in complecated samples base on their performance.Other traditional methods were adopted to further confirm the reliability of the results getting from these new developed fluorescence methods.Main contents in this dissertation are as follows: 1. Establishment of a simple and rapid method for miRNA-21 detection in tumor samples based on molecular beaconThe purpose of this chapter is to develop a new method for detecting miRNA-21 in complicated tumor samples by using target-specific molecular beacon. The characteristics of stability, melting temperature and specificity of the beacon were investigated by fluoremetry. Then, the levels of miRNA-21 in cultured tumor cells and gastric cancer tissues were detected by using the new method with optimazing reactive conditions. The results demonstrated that molecular beacon showed good performance and specificity when it was applied into standard sample detection and the limit of detection can reach 0.5 n M, Furthermore, it was used to detect the levels of miRNA-21 detection in tumor cells and tissues samples and the results indicated that the miRNA-21 level in 10 of 17 gastric cancer cases(59%) is higher than the adjacent tissues. The results of q RT-PCR also confirm it. Finally, the levels of PDCD4 and PTEN, which were regulated by miRNA-21 was detected by Western blotting and it was found that both of them negtively related with that of miRNA-21. 2. Ultrasensitive detection of miRNA-21 by combining molecular beacon with enzyme catalytic signal amplificationAn ultrasensitive method for miRNA-21 detection was established by using a system containing a chimeric molecular beacon and duplex specific nuclease. Their stability, anti-enzyme digestive capacity and hybridizing conditions of the beacon were investigated by fluoremetry. Then, we investigated the limit of detection by using the synthetic miRNA-21 as standard targets and the results show that it can reach 1 p M, which increase for about 3 orders of magnitude comparing with the method of the solely molecular beacon. Finally, the new method was used for miRNA-21 detection in 0.2 ?g total RNA of gastric cancer tissues. In summary, the new method can rapidly and reliably differentiate the difference of miRNA-21 in complicated tumor samples. 3. Real time monitoring uracil excision using uracil-containing molecular beaconThis chapter is mainly focusing on establishing a real time monitoring method of UDG analysis by using uracil-containing molecular beacon. The method was further applied for activity analysis and kinetics study of UDG. The results show that the detection limit of UDG is 0.005 U/ml, The KM and kcat are 0.89±0.1 ?M and 210±10 min-1.The 5-Fu and cisplation can inhibit UDG activity with the IC50 values of 6.1±0.52 m M and 3.2±0.24 m M. Finally, the new method could specifically detect UDG epression level in different cell-free extracts and serum. |
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