Construction And Application Of Label-free Nucleic Acid Probe For High Sensitive Nucleic Acid Detection

Nucleic acid probes as a basic detection tool for modern research in the lifesciences, compared with the general detection method, possess intrinsic advantagessuch as high sensitivity, good stability, high specificity, easy synthesis, simple designand so on. Based on the developed nucleic acid probes researchers have beencontinuously developed a series of new nucleic acid probes and detection methods,and been widely applied in fields of biology, chemistry, medical diagnosis anddisease detection etc. This paper constructed some label-free novel nucleic acidprobes based on cycled strand displacement amplification technology andexonuclease I, which have been applied to the highly sensitive nucleic acidsdetection. The detailed content is described as follows:1. A label-free novel nucleic acid probe system based on the polymerase cycledstrand displacement amplification has been developed. In this system, the nucleicacid probe can be either as a probe to identify target molecules or can be a templatefor polymerase reaction. The label-free novel nucleic acid probe system combine withstrand displacement activity of the polymerase, possess some advantages such assimple design, easy operation and low cost. Also, this system can achieve circulatingpolymerization without directly amplification of target molecules under isothermalconditions and without exogenous contamination. The nucleic acid can bequantitatively detected and analyzed by this method.2. A method for highly sensitive nucleic acids detection based on a novel nucleicacid probe-mediated signal amplification has been developed. The long-tail of thenovel probe can extend the length of the double-stranded DNA product when theprimer is annealing. Then more fluorescent dye molecules can be embedded resultingin releasing amre strong fluorescence signal. This method combined with theamplification reaction cycle. The sensitivity of the nucleic acids detection is improveby combining with the cycled amplification reaction. The limit of the nucleic acidsdetection reach to50aM. Compared with the molecular beacon probe detectionmethods, the advantage of this method is not labeled and probe-mediated thedetection signal amplification wich can further improve the sensitivity of nucleic aciddetection. 3. A novel nucleic acid probe for ultra-sensitive detection of DNA or miRNAbased on exonuclease I has been developed. Exonuclease I degrades single-strandedDNA in a3’→5’ direction. When no presence of the target molecules, exonuclease Ieffectively degrades the primers and probes from the3’-OH end, which greatlyreduces the background fluorescence signals, improves the signal to noise ratio, sothat the detection sensitivity nucleic acids the has been greatly improved. In thepresence of the target molecules, the formation of isothermal strand displacementpolymerization reaction can product a large number of long double-stranded DNAwith generating strong fluorescence signal. So that the novel nucleic acid probesachieve a PCR-like sensitivity without labeling, and the detection limitis5zM, whichreduces the six orders of magnitude compared with the general molecular beaconprobes. Furthermore, the method can detect a high similarity of miRNA fromdifferent molecules that the detection limit reaches to5zM, and the selectivity of thisapproach is excellent..