Study On Novel Rolling Circle Amplification Techniques

Rolling Circle Amplification (RCA) is an isothermal amplification technique ofnucleic acid, borrowing from rolling circle replication of pathogenic microorganisms.With the advantages of simplification and high efficiency, RCA attracts more andmore attention. It not only plays a key role in the various fields of biological science,but also has been widely used in agriculture, medicine, forensic analysis,environmental monitoring, and so on. In the first generation of RCA, when circleDNA, primer and polymerase exist in the same system, the primer will be extended toa linear single stranded DNA with short tandem repeats which are complementarywith circle DNA templates. So, the first generation RCA is known as Linear RCA(LRCA). Although, the research of RCA has been relatively mature, it still has manyproblems, such as low sensitivity, weak specificity. Recently, as a variety of methodsof isothermal amplification continue to emerge, it is a great opportunity for furtherdevelopment of RCA. Based on these backgrounds and original LRCA, we conducteda in-depth research for RCA and created new isothermal amplification techniques ofnucleic acid, which is beneficial for highly sensitive nucleic acid detection and novelbiological sensors building.Chapter1Study of a Novel Netlike Rolling Circle AmplificationIn this chapter, a novel isothermal nucleic acid amplification technique termed asNetlike Rolling Circle Amplification (NRCA) is proposed, which is based on LinearRCA(Amplification of One dimension) and Hyperbranched RCA (Amplification ofTwo dimension). We introduced a special endonuclease to improve the amplificationefficiency without the extra time and operation, and achieve the three dimensionalamplification. We use the traditional means of agarose gel electrophoresis to optimize the reaction condition of NRCA, fluorescence quantitative PCR to realize thereal-time monitoring for amplification reaction of different concentrations of targetDNA, and Atomic Force Microscopy (AFM) to intuitively characterize theamplification product. Detailed comparison of LRCA, HRCA and NRCA has beenmade, and the experimental results show that detection of target DNA can be achievedwith super-high amplification efficiency by using the third generation nucleic acidamplification technique, i.e., NRCA. The detection limit is lowered to13aM and thedetection range is extended from0.1fM to1μM. Furthermore, visualization of theproducts of NRCA has also been successfully achieved by using atomic forcemicroscopy. Surprisingly, dense and uniform network morphology is observed,making the netlike” amplification veritable not only in theory but also in practice.This is the first time to realize the netlike amplification and its visual characteration.Compared with LRCA and HRCA, NRCA owns the advantages of operability, costand time saving of original technique, and also achieves a greater signal amplification.All of theses benefits provide good technical conditions for the low abundance nucleicacid analysis and detection, which has a wide prospect for application.Chapter2Study on the Dual Signal Amplification Based on Rolling CircleAmplification and Hybridization Chain ReactionWith the optimal design of the RCA and the introduction of Hybridization ChainReaction (HCR), we constructed a simple and convenient nucleic acid amplificationtechnique－HCR@RCA. In the system of HCR, there are only two partiallycomplementary oligonucleotides which structure as hairpin. The extension process ofHCR does not require any enzymes and only can be triggered by target DNA. Whentarget DNA is present, the two hairpin probes will be opened in succession, whichleads to the polymerization of oligonucleotides into a long nicked dsDNA molecule. We ingeniously combined HCR with RCA. RCA can amplify a large number of targetDNA and every copy can trigger the HCR, so as to realize the further expansion of theRCA product. We use the traditional means of agarose gel electrophoresis to optimizeHCR@RCA reaction condition of combination, fluorescence quantitative PCR torealize the real-time monitoring of amplification reaction for different concentrationsof target DNA, and Atomic Force Microscopy (AFM) to intuitively characterize theamplification product. Compared with the traditional RCA and HCR, the results showthat HCR@RCA amplification efficiency and sensitivity are greatly improved. Inaddition, the two sequence-specific processes are both independent and integrated,and their combination is expected to futher enhance the accuracy analysis of nucleicacids. The system intergrates two independent signal amplification techniques andoffers a good reference to the design for the development of novel isothermal nucleicacid amplification technique.