The Designed, Synthesized, Appraised Identified Of Activity And Detection Of The Molecular Nucleic Acid Beacon Aptamer

The detection and quantification of molecules play an essential role in basic discovery research as well as in clinical practice. Technologies that allow specific detection and precise quantification of molecules are evolving to cater to new analyses as well as to improve existing techniques. As a result, novel approaches that challenge traditional methods are being discovered.We have designed a novel nucleic acid beacon aptamer, which can recognize target molecules with high affinity and specificity and transduction signal with high amplification potential of DNA replication in real-time quantitative PCR, for detecting a wide range of ligands. Firstly, Specific oligonucleotide aptamers to Fc fragment of mouse IgG are obtained by the systematic evolution of ligands by exponential enrichment (SELEX), the specificity of oligonucleotide aptamers to Fc fragment of mouse IgG was measured by dot-blot, high performance liquid chromatography( HPLC), Electrophoretic mobility shift assay (EMSA). As a result, 16 aptamers have been obtained. In the process, a novel SELEX selection method using nitrocellulose filter established firstly.On the basis of previous work, the molecular nucleic acid beacon aptamer was designed, synthesized and identified for the first time. Beacon sequences with fluorescence probes were linked at the 5'-end of the aptamers by nested PCR, which contain Taqman probe's complementary sequence flanked by defined primer binding sites. and is tag field to target molecules. to construct the dsDNA-labeled beacon aptamers. The construction of beacon aptamer with a dsDNA beacon has no negative effect the conformational change of aptamer on aptamer-targets recognition and binding, which facilitate aptamer-target recognition and signal transduction. In addition, the linking dsDNA beacons are also able to act as amplification templates in PCR. The activity of the beacon aptamer binding with target molecules have been demonstrated by the double method of the autoradiography and the Coomassie Brilliant Blue dyeing to agarose gel EMSA。The methods, which were nucleic acid beacon aptamer mediated immuno-IPCR (NBA-IPCR) and nucleic acid beacon aptamers mediated real-time quantitative PCR (NBA-PCR), were established for the first time. The functionality of beacon aptamer has been proved by the methods of NBA-IPCR and NBA-PCR. The beacon aptamer-IgG were used as the independence detection molecule to identify and bind the HBVAg via immunological reaction in NBA-IPCR. The signal transduction function of beacon aptamers from antigens to nucleic acid was accomplished by the compound of beacon aptamer-IgG-Ag in NBA-IPCR. The results of BA-IPCR indicate that beacon aptamers have the function of identify and bind the HBVAg, signal transduction and amplification templates in PCR. In NBA-PCR, the beacon aptamer were used as the independence detection molecule to identify and bind the target via ligand-identify reactions. The signal transduction function of beacon aptamers from target to nucleic acid was accomplished by the compound of beacon aptamer-target in NBA-PCR. The results of NBA-PCR indicate that beacon aptamers have the versatile molecular recognition of ligands and the high amplification potential of DNA replication in PCR. As a result, NBA would be able to detect target molecules by real time-PCR.In summary, due to the nucleic acid beacon aptamer has the functionality of identification ligand, signal transduction and the high amplification potential of DNA replication in PCR, which is a novel molecule with flexible construction, high specificity and sensitivity, and would be able to detect various target molecules.