Construction Of Cascade Amplification System By Exonuclease Ⅲ-assisted Target-recycling And Its Nucleic Acid Detection

As an important endogenous regulatory molecule,nucleic acid is closely related to the occurrence,development and metastasis of diseases.Therefore,as an ideal biomarker,nucleic acid is of great significance in the early diagnosis and postoperative monitoring of tumors.At present,the traditional methods of nucleic acid detection include gel electrophoresis,isotope labeling and polymerase chain reaction(PCR).Because of their low sensitivity,complex operation and high cost,they are widely used.In addition,in the early stage of tumor,the expression of nucleic acid is very low,so it is very important to develop new methods for high sensitive detection of nucleic acid.Exo-Ⅲ is a kind of nucleic acid hydrolase,it has the advantages of high sensitivity and specificity.Based on this,in order to develop new methods with high sensitivity and specificity for nucleic acid detection,two cascade amplification strategies based on Exo-Ⅲ assisted target cycle were developed by using Exo-Ⅲ’s ability to achieve target cycle amplification and combining the signal amplification strategies of hybridization chain reaction(HCR)and DNAzyme,Taking p53 gene and miRNA as the research objects,we carried out the detection of disease-related nucleic acid markers,and investigated their detection ability in serum samples and cell culture medium.The specific research contents are as follows:1.Detection of DNA by Exonuclease Ⅲ-Assisted Target-Recycling and HCR Cascade AmplificationA simple and isothermal cascade signal amplification strategy for nucleic acid detection was successfully developed by using Exo-Ⅲ assisted target cycle amplification technology and HCR signal amplification technology.The method consists of two steps.The first step is composed of trigger probe trigger and auxiliary probe L,which is used to realize the target cycle amplification;The second step consists of hairpin probes H1 and H2 which trigger HCR to amplify the signal.When the target exists,the target can hybridize with the free sequence of the auxiliary probe in the hybrid double chain of the trigger probe and auxiliary probe L to form a double chain structure with 3’ end flush.Under the hydrolysis of Exo-Ⅲ,the auxiliary probe can be cleaved to release the target and the trigger,and the released target can continue to participate in the next cycle to produce more trigger sequences;The released trigger chain trigger can participate in the second step reaction,trigger HCR reaction,and realize signal amplification.Taking the target p53 gene as the research object,the experimental results show that the sensing strategy can realize the sensitive detection of the target p53 gene,and the detection limit is as low as 2.0 p M.At the same time,this method can also realize the detection of target in serum,with good specificity and stability,which is expected to provide a new opportunity for clinical sample research.2.High Sensitive Detection of miRNA by Exonuclease Ⅲ-Assisted Target-Recycling and DNAzyme Cascade AmplificationIn order to further improve the sensitivity of the detection method,we introduced DNAzyme(P1)signal amplification technology and developed a cascade signal amplification method to achieve high sensitivity detection of target miRNA by using Exo-Ⅲ assisted target cycle amplification capability.The method consists of two steps: cycle I is composed of DNAzyme and auxiliary probe P2 to achieve the target cycle amplification;Cycle II consists of a report probe(MB)containing RNA sites for signal amplification.First,in cycle I,when the target does not exist,DNAzyme hybridizes with the auxiliary probe(P2)to form a double stranded structure.At this time,part of the free sequence of P2 is leaked.When the target exists,it hybridizes with the free sequence of auxiliary chain P2 to form a double chain structure with 3’end flush.The target and DNAzyme are released through Exo-Ⅲ hydrolysis.The released target can participate in the next round of target cycle and generate more trigger sequences;The released DNAzyme can participate in the second step cycle reaction.With the assistance of zinc ion,it continuously cuts the substrate chain,leading to the separation of fluorescent group and quenching group,thus achieving fluorescence amplification.The experimental results show that the method can achieve high sensitivity detection of miRNA-21,and the detection limit is as low as0.19 f M.At the same time,the method has high selectivity.In addition,this method can also be used for the sensitive detection of targets in cell pyrolysate,which is expected to provide a new method and technology for the highly sensitive detection of tumor markers.