High Sensitive Gene Detection Based On EXOⅢ-assisted Fluorescence Signal Amplification Probe And Gel Electrophoresis-based Selection Of Aptamers Against Cells

In the first part of this paper, we have studied the signal amplification technology and its application in gene detection. At first, the Fe₃O₄ magnetic beads were synthesised with activated PAA（MW=8000）. The fluorescent nucleic acid probe was fixed on the surface of Fe₃O₄ magnetic beads by amino and carboxy reaction to prepare fluorescent nucleic acid probe-Fe₃O₄ magnetic beads complexes. Fe₃O₄ magnetic beads can not only as a separate carrier, but also can effectively quench the fluorescent group of fluorescent nucleic acid probe, which reduced the background fluorescence signal and laid the foundation for the subsequent fluorescent signal amplification.After the addition of the target molecule, a double chain was formed by the base complementation between target molecules and fluorescent nucleic acid probes. The unique hydrolysis characteristics of exonuclease EXOIII, which made the EXOIII hydrolysis probe, release the target molecule and the fluorescence signal. Then the target molecules continued to hybrid with excess fluorescent nucleic acid probe and triggered EXOIII-assisted target molecular cycle reaction. According to this, an EXOIII-based approach for DNA detection was developed. Compared with the traditional molecular beacon method, this method had higher sensitivity, and the ability of fluorescence signal amplification was more significant. Moreover, the gene SNP can be rapidly genotyping by the different fluorescence detection signal from mixed fluorescent probes with double fluorescent labeled.In recent years, an increasing number of nucleic acid probes are used for the early diagnosis and treatment of cancers by identifying and detecting cancer cells. Aptamers are single-stranded DNA / RNA probes with properties similar to antibodies, which can be widely used in the field of molecular biology and medicine as molecular probes. The second part of this paper was based on agarose gel electrophoresis technology, and combined with the cell-SELEX（cell-based systematic evolution of ligands by exponential enrichment） to select aptamers against intact cells. HL-60 cells were acute promyelocytic leukemiacells, which were served as target cells. The target cells were fixed in agarose gel, and the aptamers with strong specific to target cells were separated by the difference of electrophoretic mobility during electrophoresis. By optimizing electrophoresis, PCR and other conditions, panels of aptamers against HL-60 cells have been evolved from aninitial DNA library by five screening.