The Activity Of Nucleic Acid Tools Was Determined By SYBR Green I As A Probe

In view of its analytical advantages of high sensitivity and rapid response time, fluorescent biosensing technology has been widely used in the detection of various biomolecules. Traditional fluorescent biological analysis usually requires fluorophore labeling which might add cost and complexity for the assay. Therefore, it is of great importance to develop some unlabeled fluorescence biological analysis methods so as to enhance the bioanalytical performance and reduce the cost. This thesis developed some novel and label-free fluorescent biological analysis methods for detecting the activity of two tool enzymes based on a signal probe SYBR Green I. The details are described as following two chapters:1. Label-free fluorescence strategy for exonuclease Ⅲ activity detection using SYBR Green I as probeA label-free and sensitive fluorescence assay for exonuclease Ⅲ activity is developed based on the phenomenon that SYBR Green I (SG) can specifically bind the double-stranded DNA, and make its fluorescence intensity greatly improved. Two completely complementary ssDNA would hybridize to form stable DNA duplexes. With SG selective staining of the DNA duplexs, a high fluorescence signal was obtained. Upon the addition of Exo Ⅲ, the stable DNA duplexes would be digested into mononucleotides, a weak fluorescence signal was observed due to the weak interaction between SG and mononucleotides. So Exo Ⅲ activity can be facilely measured with a simple fluorescence reader. The feasibility of this strategy has been further verified by circular dichroism (CD) spectroscopy analysis. The process of deactivation of Exo Ⅲ was also investigated in the assay. Therefore, it is a simple, quick and cost-effective fluorescence method for detection of Exo Ⅲ activity without any complex DNA sequence design or fluorescence dye label.2. Label-free fluorescent method for DNA Polymerase I activity detection based on graphene oxide-assisted background reducingA novel and label-free fluorescence assay for detection of the activity and inhibitors of DNA Polymerase I was developed based on the unique absorbing ability and universal quenching properties of graphene oxide (GO) and DNA elongation reaction. DNA Polymerase I (KF) can selectively catalyse substrate dNTP along the primer 5’ to 3’ direction continuous polymerization, and form a long double-stranded DNA structure whose sequence is completely complementary with template sequence. And then the SG can quickly bind the long double-stranded DNA structure resulting in the strong fluorescent signal. When no KF polymerase exsited in the system, polymerase elongation reaction can not happen successfully and double-stranded DNA structure was not produced in the system, which resulted in the weak fluorescent signal when SG was added into the system. So we can study DNA polymerase KF activity and inhibition of its inhibitor based on the changes of the fluorescence intensity of the system in the prescence or absence of DNA polymerase KF. The introducion of GO improved the signal to noise ratio of the system and improved the sensitivity of our method. Therefore, it is a label-free, cost-effective, sensitive fluorescent method for detecting DNA polymerase KF activity.