| Nucleic acid is one of the most important biomolecules in life that is in charge of transferring genetic information and coding other biomolecules. The investigation of nucleic acids has been one of the most important areas and currently one of the most rapidly growing fields in contemporary molecular biology and biotechnology.Nucleic acids ligation catalyzed by DNA ligase plays an indispensable role in cell life cycle, which includes many essential life processes such as replication, repair and recombination of nucleic acids. Unfortunately, up to now, our knowledge of interactions between ligase and nucleic acids has been limited because of the lack of adequate methodologies for real-time detection. The detailed and fundamental nature of these interactions as well as their contribution to the total organization of biological systems is yet to be discovered. Better understanding of these interactions not only will benefit the understanding of many biological mechanisms but also is expected to be of great medical use in designing new drugs and in developing new disease treatment strategies.Detection of virus genome RNA is of great importance for diagnosis of some diseases infected by RNA virus. RT-PCR, commonly used for RNA virus determination, requires template of high quality, which has restricted application of the method. Therefore, it is necessary to develop technology for quick and sensitive detection of virus RNA.Molecular beacons are a class of novel fluorescence probes, since first developed in 1996, they have been widely used in DNA/RNA and some protein studies. Molecular beacon has shown to be a useful tool in monitoring interactions between nucleic acids and proteins (enzymes), which makes it a pretty good tool for monitoring interactions between proteins and nucleic acids in real time.Furthermore, since molecular beacon is a kind of probe with high sensitivity, high selectivity, low background and no need of remove from the redundant probes from detecting mixture, it could be used to determine mRNA and genome RNA of somevirus without amplification by PCR.Based on molecular beacon probes, Dr. Zhiwen Tang in our lab has developed novel methods to research on ligation, phosphorylation and enzymatic cleavage of nucleic acids. As a part of his systematic research, the thesis has developed application systems based on molecular beacon for two goals: one is to monitor reactions between nucleic acids and proteins (enzymes) in real-time, the other is to detect virus RNA quickly and directly. The thesis is composed of three major projects:(1) Real time monitoring of nucleic acids ligation catalyzed by T4 DNA ligaseand foundation of kinetics model of the methodA new approach has been developed for monitoring DNA ligation using molecular beacon (MB) DNA probes. Molecular beacon is a hairpin-shaped oligonucleotide probe with high sensitivity and specificity. It has been employed as a DNA probe to monitor the ligation process. The sequence of the MB was designed to be complimentary with the product of the ligation, therefore, the ligation process can be monitored in a homogeneous solution in real-time. It could be realized based on this method to study nucleic acids ligation kinetics with convenience and to determine the activity of DNA ligase accurately. The result of kinetics experiment indicates that interaction in the detecting system is a single-substrate enzyme-catalyzed reaction and molecular beacon acts as not only the probe for monitoring ligation but also the template for DNA substrate formation. Effects of some external factors and base mismatches at the nick on DNA ligation have been studied using the novel approach. Based on this principle, a sensitive, precise and quick analytical method to determine the activity of T4 DNA ligase has also been developed.The major advantages of our method are its ultrasensitivity, excellent specificity, convenience, real-time monitoring of homogeneous solution. The now easily obtainable MBs would found wide application in the investigation o...
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