Development And Biomedical Application Of Rolling Circle Amplification Technology

Nucleic acid isothermal amplification is a process in which nucleic acid is spontaneously and efficiently amplified with a certain sequence template at a constant temperature,thereby obtaining a large number of nucleic acid fragments with repetitive sequences.This technology does not require thermal cycling equipment which is efficient,fast,easy to operate,highly sensitive and highly specific,etc.In recent years,researches on various nucleic acid isothermal amplification techniques have been emerging,especially in nucleic acid detection,showing broad application prospects.At present,a variety of nucleic acid isothermal amplification techniques,including loop-mediated isothermal amplification,DNA strand displacement amplification,nicking enzyme amplification reaction and rolling circle amplification,etc.,have shown great advantages in the convenience and timeliness of detection.Based on the above background,in this paper,we have carried out in-depth development and research on rolling circle amplification technology.On the basis of the original work,through careful design,a new isothermal nucleic acid amplification technology with better performance has been developed to provide conditions for the highly sensitive detection of nucleic acids and the construction of new biosensors.The thesis work mainly includes the following two aspects:1.NRCA-based paper sensor for visual ultrasensitive detection of SARS-Co V-2In this chapter,on the basis of the existing netlike rolling circle amplification(NRCA),we use the program design of nucleic acid sequences,the introduction of padlock probes,the optimization of reaction system and other conditions,and real-time quantitative PCR to target different concentrations of targets.The DNA amplification reaction was monitored in real time,and the amplification products of the NRCA reaction were visually characterized by atomic force microscopy.The technical application of NRCA in the nucleic acid detection of SARS-Co V-2 was developed,reaching a detection limit of 4.12 a M.After the padlock probe is combined with the target nucleic acid,the padlock probe is circularized by the action of T4 ligase,thereby triggering the NRCA reaction in the nucleic acid amplification system to achieve signal amplification.Through the observation of electrophoretic bands,this method exhibits excellent specificity,and the direct observation results of AFM show that the shape of the amplified product of NRCA presents a dense and uniform network structure,thus confirming this highly efficient "netlike" amplification in practice.We also applied the reaction system to the paper sensor.The paper sensor is made by spraying the reaction system and pullulan solution on the nitrocellulose membrane through inkjet technology.The sensor can intuitively characterize the target under the fluorescence imaging system,and the fluorescence signals of NRCA products at different target concentrations can be observed after loading the sample.The results show that this method can ensure the performance of high efficiency,high sensitivity,high specificity and visualization,etc.,while achieving the low cost of SARS-Co V-2 nucleic acid detection and the need for no thermal cycling equipment.The further popularization of the epidemic and the comprehensive prevention and control of the epidemic provide the possibility.2.Quantitative analysis and gene identification of ec DNA based on CRIRCAIn this chapter,we combine the ingenious design of netlike rolling circle amplification(NRCA)technology with CRISPR technology,which has recently gained prominence in gene editing technology,to develop a clinical analysis of extrachromosomal DNA(ec DNA).The new CRIRCA fusion technology(CRISPR-Cas9 nickase-mediated rolling circle amplification technology)needs to systematically study its mechanism and performance,and make it available in single cells after combining with oncogene primer library,electrophoresis and fluorescence analysis technology.The structural,sequence and quantitative information of ec DNA is obtained simultaneously at the horizontal level.The results show that this method has a more efficient signal amplification effect than the traditional RCA method,and can perform sensitive analysis of a small amount of ec DNA in tumor cells.Quantitative analysis showed that the ec DNA content of cancer cells was much larger than that of normal cells,and there were some differences in ec DNA content between cancer cells.Computer-aided design,construct ec DNA oncogene primer library and sg RNA library,design specific oncogene probes,and CRIRCA technology for identifying and analyzing ec DNA oncogenes were used in breast cancer cells.This work will fill the current gap in early cancer research in targeting ec DNA,and provide support for the precise diagnosis and treatment of cancer.