Application Of Nucleic Acid Constant Temperature Amplification Technology In Polynucleotide Kinase And MicroRNA Analysis

Protein kinase and MicroRNA all plays a crucial role of regulatory in the process of life. Their abnormal patterns are closely connected with many serious diseases, such as cancer, diabetes, heart disease, ect. Hence it has a great significance to detect the kinase and MicroRNA with a highly sensitivity for clnical diagosis, drug screening and target therapy. In this thesis, we respectively focused on microRNA and T4 PNK as research object and developed two kinds of simple nucleic acid isothermal amplification methods, which achieved the highly sensitivity detection to targets. The main contents are as follows:1.Phosphorylation-induced hybridization chain reaction on beads:an ultrasensi-tive flow cytometric assay for the detection of T4 polynucleotide kinase activityA versatile flow cytometric bead assay (CBA) is developed for sensitive PNK detection,coupled λ exonuclease cleavage reaction, by integrating the advantages of hybridization chain reaction (HCR) for enzyme-free signal amplification, flow cytom-etry for robust and rapid signal readout as well as magnetic beads (MBs) for facile separation. In this assay, a biotinylated target DNA is firstly immobilized on strep-tavidin-functionalized MBs, then, a complementary sequence was added in the solution to form dsDNA. Afterwards. dsDNA is phosphorylated by PNK and then immediately cleaved by λ exonuclease, each target would initiate a cascade of hybriddization events between two species of fluorescent DNA hairpin probes (H1*/H2*) to form a nicked double helical DNA structure, resulting in amplified accum-ulation of numerous fluorophores on the MBs. Finally, the MBs are directly analyzed by flow cytometry. The detection limite of this methods toward PNK was obtained as 1×10-5U/mL (3a), which is 2 orders of magnitude lower compared with reported methods. Furthermore, this method can also be used to screen the inhibition effects toward several common inhibitors. Moreover, this strategy is also successfully applied to the PNK detection in complex biological samples, showing great potential for biological process research, drug discovery, and clinic diagnostics.2.Novel Fluorescent Biosensor for Sensitive Detection of MicroRNA Based on Duplex-Specific Nuclease(DSN) Coupled with Terminal Deoxynucleotidyl Trans- ferase(TdT)Based on Duplex-specific nuclease(DSN) and Terminal deoxynucleotidyl transferase(TdT), we have established a novel fluorescent biosensor for sensitive and selective detection of MicroRNA. DSN shows a strong preference for cleaving double-stranded (ds) DNA and DNA in DNA-RNA hybrid duplexes, TdT is an enzyme that catalyzes the repetitive addition of mononucleotides from dNTPs to the terminal 3’-OH of a DNA initiator. In the presence of MicroRNA,the DSN would cleaving DNA hybridization with RNA, then the RNA would hybridize with another DNA. This could form the first step of amplification. In the second step of applification, these cleaved oligonucleotides would be added thousand of repetitive mononucleotides on the 3’ terminal. Based on these two steps of amplification, the detection limit is as low as 500fM. MicroRNA plays an important role in gene controlling and has complex relationship with many diseases. So this strategy shows great potential for biological process research, drug discovery, and clinic diagnostics.