Detection of DNA Target with Highly Enhanced Specificity by Self-Circularization Rolling Circle Amplification
DNA detection is widely used for gene analysis. However, false-positive results are difficult to be avoided due to the non-specific amplification. Here, we described a novel RCA approach to improve the specificity. With the help of TspR I (endonuclease),
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Detection of DNA Target with Highly Enhanced Specificity by SelfCircularization Rolling Circle Amplification Xingyu Wang, Xiaoliang Wang, Ping Dong, Masatomo Suzuki, Hiroyuki Asanuma and Xingguo Liang Abstract DNA detection is widely used for gene analysis. However, false-positive results are difficult to be avoided due to the non-specific amplification. Here, we described a novel RCA approach to improve the specificity. With the help of TspR I (endonuclease), DNA was cleaved into smaller duplex fragments, including the target DNA fragment, with 9 nt sticky ends. A duplex adaptor with two sticky ends which were complementary to that of the target fragment was designed. After hybridization of the adaptor with the target fragments, T4 DNA ligase was used to ligate them to form a circular ds DNA with a gap. Then RCA was carried out from the free 30 -end of the open strand by Phi29 DNA polymerase with two primers which were complementary to the target sequence. Different from the traditional padlock-RCA, SC-RCA showed excellent specificity by amplifying the specific DNA target other than the added probe. Keywords RCA
DNA detection DNA amplification Gene analysis
Xingyu Wang and Xiaoliang Wang—these authors contributed equally to this article. X. Wang X. Wang P. Dong X. Liang (&) College of Food Science and Engineering, Ocean University of China, Yushan Road, No.5, Shinan-qu, Qingdao 266003, People’s Republic of China e-mail: [email protected] M. Suzuki H. Asanuma Department of Molecular Design and Engineering, Graduate School of Engineering, Nagoya University, Chikusa, Nagoya 464-8603, Japan
T.-C. Zhang et al. (eds.), Proceedings of the 2012 International Conference on Applied Biotechnology (ICAB 2012), Lecture Notes in Electrical Engineering 251, DOI: 10.1007/978-3-642-37925-3_154, Ó Springer-Verlag Berlin Heidelberg 2014
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154.1 Introduction DNA amplification technology has greatly facilitated the development of molecular biology. Based on the amplification protocols, nucleic acid detection for harmful bacteria and virus won the edge in diagnosis, infectious disease prevention, environment monitor, and food industry [1]. As a traditional amplification approach, polymerase chain reaction (PCR) has been widely used for detecting DNA targets with high specificity and sensitivity. However, besides the elaborate routine for primer design, since PCR requires expensive equipment for fast thermal cycles and precise temperature control, its application in low-cost and rapid DNA detection has been limited [2]. Also, PCR can lead to sequence-dependent bias due to the mismatched primer-target hybridization and the intense thermal cycles [3]. As an isothermal detection method for overcoming the shortcomings of PCR, RCA (rolling circle amplification) has gained a great attention over the past decade [4]. The most distinguished feature of RCA is that it can be easily carried out on a chip for high-throughput detections [5]. However, the specificity of traditional padlock-RCA is not high
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