Study On The DNA Amplification Technology Of Self Circularization-Rolling Circle Amplification At Ambient Temperature

The amplification technology of specific nucleic acid sequence has been a basictechnology in Molecular biology. The technology used for amplification of specificnucleic acid sequence can be divided into two categories, temperature cyclicamplification and isothermal amplification.DNA cloning technology is a kind of DNA amplification technology whichneeds the aid of Escherichia coli host bacteria, so its operation is complicated in theapplication of detection. PCR technology is a kind of rapid in vitro DNAamplification technology, however, it must need high speed variable temperaturecontrol and require a specific apparatus. Especially in food safety monitoring, a largenumber of samples processing simultaneously, the need for high-throughputprocessing, rapid amplification of DNA, high cost, low efficiency, it is difficult toachieve the practical application.Isothermal DNA amplification technique has great advantage for rapid detection;it does not need special temperature control equipment and can generate largeamplification in a short time. The LAMP technique is a simple and efficient DNAamplification technology, but once non-specific product is generated, it cannot befound in time. It affects the technology of detection specificity. In addition, the primerdesign of the technique is complicated. Comparing with LAMP technology, Rollingcircle amplification is a kind of isothermal DNA amplification technology, the currentmature RCA method is mainly padlock probe RCA (Padlock-RCA). This method isonly a signal amplification method, the hybridization of probe with the template isoften wrong and produces non-specific amplification, and long probe synthesis cost isalso relatively high.The Self circularization-RCA studied in this paper is a kind of improved rolling circle amplification technology. Self circularization-RCA utilizes a restrictionendonuclease (TspR I) which has a specific enzyme cutting site digest targets of DNA,through the DNA Ligase specifically connect the fragment with the adaptor which isthrough specifically design to complemented to the former, with the phi29DNApolymerase amplification, achieve the goal of DNA rolling circle replication. Thehigh cost of long padlock probe synthesis is saved, and the sticky ends with9basepairs can ensure the specificity of circularization. SC-RCA technology avoids theproblem that the distinction of non-specific amplification is insufficient which oftenexist in the LAMP technology, and its circularization is simple comparing with thePadlock-RCA, and not only is the signal amplification, is also a kind of templateamplification. SC-RCA has very broad application prospects in the nucleic acid rapiddetection field.This paper mainly studies the SC-RCA technology, including the experimentalconditions of specificity, the sensitivity experiments and some explorations aboutsimplizing the operation of the technology.We study the specific experimental conditions of SC-RCA, T4DNA Ligasespecific ligation conditions are as follows:40oC for10min, then slowly cooled to25oC temperature, after a short time (20min) ligation, specifically to generate circulartemplate. Taq DNA Ligase is a heat-resistant DNA Ligase, its specific ligationconditions are as follows:45oC for10h, specifically to generate circular template.The experimental results show that, the key experimental factors of the SC-RCAreaction specificity mainly concentrate on the ligation and the use of primer inamplification. The mainly influential conditions in ligation are reaction temperatureand suitable ligase. The specific condition of the ligation in SC-RCA is that reactionat45oC for10h through Taq DNA Ligase.Using research to specific connection of experimental conditions for sensitivityexperiments, simple fragment can be amplified for minimum template concentrationwas1pM, enzyme digestion of plasmid multiple fragments can be amplified under thecondition of minimum template concentration of10fM. We will also compareSC-RCA technology with PCR technology, PCR technology can be amplified specific fragments from100aM plasmid templates, sensitivity is higher than that in SC-RCAtechnology, but mixed with other genomic cases, PCR technology is easy to producenon-specific amplification, and SC-RCA technology can be used in such complicatedconditions to amplify specific template fragments.We also explore the application of SC-RCA technology. The restriction effect ofTspR I restriction endonucleases is good in the Taq DNA Ligase reaction buffer, for358bp products,500ng can be completely cleaved by1U restriction endonuclease at45oC for40min, therefore, restriction endonuclease interference with the effect ofsingle tube reaction. SC-RCA reaction on Solid beads, using an adaptor fixed on thebeads, and then exerts ligation and amplification on the beads. Experimental resultsshow that the amplification of specific fragment on solid beads is more difficult thanthat in liquid phase, we first conducted in a liquid phase connection response,subsequent amplification on a solid phase, can be amplified product. Preliminaryexperimental results show that the circularization on solid-phase beads is difficultrelative to the liquid phase.