Cut-and-Paste of DNA Using an Artificial Restriction DNA Cutter.docVIP

Int. J. Mol. Sci. 2013, 14, 3343-3357; doi:10.3390/ijmOPEN ACCESS International Journal of Molecular Sciences ISSN 1422-0067 /journal/ijms Review Cut-and-Paste of DNA Using an Artificial Restriction DNA Cutter Makoto Komiyama Life Science Center of Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan; E-Mail: komiyama@tara.tsukuba.ac.jp Received: 5 December 2012; in revised form: 28 January 2013 / Accepted: 30 January 2013 / Published: 5 February 2013 Abstract: DNA manipulations using a completely chemistry-based DNA cutter (ARCUT) have been reviewed. This cutter, recently developed by the authors, is composed of Ce(IV)/EDTA complex and two strands of pseudo-complementary peptide nucleic acid. The site-selective scission proceeds via hydrolysis of targeted phosphodiester linkages, so that the resultant scission fragments can be easily ligated with other fragments by using DNA ligase. Importantly, scission-site and site-specificity of the cutter are freely tuned in terms of the Watson–Crick rule. Thus, when one should like to manipulate DNA according to the need, he or she does not have to think about (1) whether appropriate “restriction enzyme sites” exist near the manipulation site and (2) whether the site-specificity of the restriction enzymes, if any, are sufficient to cut only the aimed position without chopping the DNA at non-targeted sites. Even the human genome can be manipulated, since ARCUT can cut the genome at only one predetermined site. Furthermore, the cutter is useful to promote homologous recombination in human cells, converting a site to desired sequence. The ARCUT-based DNA manipulation should be promising for versatile applications. Keywords: site-selective scission; DNA cutter; Ce(IV)/EDTA; PNA; human genome; homologous recombination; restriction enzyme-free manipulation 1. Introduction Current biotechnology is primarily based