Enzyme Treatment-Free and Ligation-Independent Cloning Using Caged Primers in Polymerase Chain Reactions.docVIP

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Enzyme Treatment-Free and Ligation-Independent Cloning Using Caged Primers in Polymerase Chain Reactions.doc

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Enzyme Treatment-Free and Ligation-Independent Cloning Using Caged Primers in Polymerase Chain Reactions

Molecules 2012, 17, 328-340; doi:10.3390/moleculeOPEN ACCESS molecules ISSN 1420-3049 /journal/molecules Article Enzyme Treatment-Free and Ligation-Independent Cloning Using Caged Primers in Polymerase Chain Reactions Akinori Kuzuya 1,2,*, Keita Tanaka 1, Hitoshi Katada 1 and Makoto Komiyama 1,* 1 Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904, Japan; E-Mails: keita@mkomi.rcast.u-tokyo.ac.jp (K.T.); katada@mkomi.rcast.u-tokyo.ac.jp (H.K.) 2 Department of Chemistry and Materials Engineering, Kansai University, 3-3-35 Yamate-cho, Suita, Osaka 564-8680, Japan * Author to whom correspondence should be addressed; E-Mails: kuzuya@kansai-u.ac.jp (A.K.); komiyama@mkomi.rcast.u-tokyo.ac.jp (M.K.); Tel.: +81-6-6368-0829 (A.K.); Fax: +81-6-6368-0829 (A.K.); Tel.: +81-3-5452-5200 (M.K.); Fax: +81-3-5452-5209 (M.K.). Received: 28 October 2011; in revised form: 26 December 2011 / Accepted: 27 December 2011 / Published: 30 December 2011 Abstract: A new simple scheme for constructing recombinant vectors that does not require any restriction enzyme, ligase, or any other special enzyme treatment has been developed. By using caged primers in PCR, unnatural sticky-ends of any sequence, which are sufficiently long for ligation-independent cloning (LIC), are directly prepared on the product after a brief UVA irradiation. Target genes and vectors amplified by this light-assisted cohesive-ending (LACE) PCR join together in the desired arrangement in a simple mixture of them, tightly enough to be repaired and ligated in competent cells. Keywords: DNA; PCR; caged compounds; ligation; cloning 1. Introduction In current molecular biology and biotechnology, vectors are constructed by digesting plasmid DNA with restriction enzymes, followed by connection of this vector with predetermined gene fragment using ligase. In 1990, Aslanidis and de Jong proposed a system that may dramatically sim