Cycle Amplification Detection Of DNA And Tumor Cells Based On Nicking Endonuclease And RNA Ligase

The sensitive and selective detection of nucleic acids is important inbiological studies, clinical diagnostics since they are routinely used asbiomarkers to help diagnose pathogenic infections and genetic disorders.Specific nucleic acids indicating the presence of a disease are often foundin only trace amounts in a complex biological extract, it is necessary todevelop amplification techniques that enable the detection of trace levelsof a specific sequence. In the present study, cycle amplification detectionof DNA and tumor cells based on nicking endonuclease and RNA ligasewas investigated. The strategies result in ultrahigh sensitivity, holdinggreat promise in providing a universal sensing platform for the detectionof various targets.In the first chapter, the construction and classification of DNA biosensor were introduced. Research applications for some typicalnanoparticles, endonuclease and DNA logic gates were simply introduced.In the second chapter, a three-input DNA logic gates with the cycleisothermal amplification based on nicking endonuclease （NEase） aredesigned. An AND gate is constructed by employing DNA sequences G1and G2. There are two stem-loop structures from the self-hybridization ofG1and one DNA bulge structure from the hybridization of G1and G2.The results of the PAGE suggest that the logic gate is formed andperforms as expected. Very low concentrations of the analytes weresufficient to initiate an autocatalytic cascade, achieving a significantimprovement of the detection limit,100-fold improvement compared tothe non-autocatalytic system. This was achieved by engineering a simpleand flexible biological circuit designed to initiate a cascade of events todetect and amplify a specific DNA sequence. The activation of the systemfor2h enzyme cycle reaction at37oC enables the detection of the targetsin the range of1.0×10^-12M to5.0×10^-11M with a detection limit of 1.2×10^-12M. This procedure has the potential to greatly simplify the logicoperation because amplification can be performed in “one-pot”.In the third chapter, by using DNA strands comprising NEaserecognition sites as inputs, an OR gate designated cascade isothermalamplification is constructed. The circuit consists of three cascades thatoperate in series. The first cascade is a three-input logic gate, which iscomposed of two DNA sequences. The second cascade is designed toinitiate isothermal DNA amplification based on nicking endonuclease. Thethird one is designed as the amplification by DNAzyme and signaltransduction. The OR gate is constructed by employing DNA sequencesG1and G2. There are three stem-loop structures, two from theself-hybridization of G1and one from the hybridization of G1and G2. Bydefinition, the logic gate OR（A,B,C） is at the “ON” state when any of theinputs is in the high state, and the output is “OFF” when all the inputs arein the low state. When the three inputs are present, they bind to theirrespective complementary loop sequences in the gate, causing the formation of G1/I1/I2complex and G2/I3hybrid. With the presence ofNb.BbvCI NEase, G1is cleaved into three fragments1,2and3, and G2iscleaved into4and5. Target sequences spontaneously dissociate from thehybrid, respectively, at the present temperature. The released I1, I2and I3then hybridize with another G1/G2logic gate and initiate a new cycle ofscission. The system enables the detection of the targets a, b and c in therange of1.0×10^-12M to5.0×10^-11M with a detection limit of2.0×10^-12M.In the fourth chapter, enzyme-free amplified detection of tumor cellbased on exponential amplification of self-sustained replication ofRNAzyme was carried out. Using carboxylated modified beads as carrier,on which amino modified Ramos cell aptamer DNA （S1） was attached.Complementary sequence S2was linked to the surface of beads throughhybridization. When joining a different number of cells in the supernatant,isolate different numbers of the S2chain. While the S2chain with RNAligase action, through the interaction of A1A2E1E2with A1A1connected into a chain of E0, generated E0chain and issuing DNA chainS3complementary, thereby S3chain is opened, fluorescence enhancement,different number of cells corresponding to different lighting value, toachieve the detection of cells for the purpose of.In the last chapter, it was summarized for the whole thesis.