Chemiluminescence Imaging For The Detection Of DNA Single-nucleotide And Multiple MicroRNAs Based On Tool Enzyme-assisted Signal Amplification

Nucleic acids are one of the expressing substances of basic life, which can be usedto diagnose diseases by detecting expressed RNA and gene mutation. This paper inview of the detection of DNA single-base mismatch and multicomponent microRNAcarried out the following two aspects of work.1. Studies on the detection of DNA single-nucleotide based on the ligase chainreaction and chemiluminescence resonance energy transferBased on ligase chain reaction (LCR) amplification, a simple and efficient methodwas developed for the detection of DNA single-nucleotide. The system consisted offour probes, which were modified with biotin, fluorescein, hemin aptamer. Upon theaddition of single-base mismatched DNA, the LCR was initiated in the presence ofampligase. The resulting products were captured by streptavidin-coated magneticbeads, which can efficiently reduce the background. Based on luminol-H2O2-DNAzyme-fluorescein chemiluminescence resonance energy transfer (CRET)system, this method achieved sensitive detection of DNA single-nucleotide bychemiluminescence imaging analysis. In addition, the method was highly selective,which can distinguish single-mismatched DNAand wide-type DNA.2. Study on chemiluminescence imaging for simultaneous detection of multiplemicroRNAs based on DNAmachine amplificationA efficient separation and highly sensitive detection of multiple microRNAs wasdeveloped based on stem-loop probe-assisted DNA machine. Target microRNAs canopen corresponding stem-loop probes that were immobilized on magnetic beads. Withthe assistance of Klenow exo-DNA polymerase and Nb.BbvCI nicking endonuclease,the DNA machine was initiated. As a result, a large amount of HRP-labled primerswere introduced onto the surface of magnetic beads. Through further stranddisplacement reaction, this method achieved chemiluminescence imaging forsimultaneous detection of multiple microRNAs by luminol-H2O2enhanced by PIPThe method had the advantages of high sensitivity, wide linear range, goodspecificity, easy operation, providing a new approach for genes expression andmolecular diagnostics of miRNAs, especially for cancer diagnosis at the early stage.