Nucleic Acids Research Advance Access first published online on September 8, 2006
This version published online on September 28, 2006
Nucleic Acids Research, doi:10.1093/nar/gkl625
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© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Chemistry |
Dynamic assembly of primers on nucleic acid templates
1 Foundation for Applied Molecular Evolution, Ayers Medical Plaza Suite 208, 720 SW 2nd Avenue, Gainesville FL 32601, USA 2 Firebird Biomolecular Sciences, Inc. Gainesville Technology Enterprise Center, 2153 Hawthorne Road, Gainesville, FL 32641-7553, USA 3 The Westheimer Institute for Science and Technology, Ayers Medical Plaza Suite 200, 720 SW 2nd Avenue, Gainesville FL 32601, USA
*To whom correspondence should be addressed. Tel: +1 352 271 7005; Fax: +1 352 271 7076; Email: nleal@ffame.org
Received June 15, 2006. Revised August 7, 2006. Accepted August 8, 2006.
A strategy is presented that uses dynamic equlibria to assemble in situ composite DNA polymerase primers, having lengths of 14 or 16 nt, from DNA fragments that are 6 or 8 nt in length. In this implementation, the fragments are transiently joined under conditions of dynamic equilibrium by an imine linker, which has a dissociation constant of 
1 µM. If a polymerase is able to extend the composite, but not the fragments, it is possible to prime the synthesis of a target DNA molecule under conditions where two useful specificities are combined: (i) single nucleotide discrimination that is characteristic of short oligonucleotide duplexes (four to six nucleobase pairs in length), which effectively excludes single mismatches, and (ii) an overall specificity of priming that is characteristic of long (14 to 16mers) oligonucleotides, potentially unique within a genome. We report here the screening of a series of polymerases that combine an ability not to accept short primer fragments with an ability to accept the long composite primer held together by an unnatural imine linkage. Several polymerases were found that achieve this combination, permitting the implementation of the dynamic combinatorial chemical strategy.
*Correspondence may also be addressed to Steven A. Benner. Tel: +1 352 271 7005; Fax: +1 352 271 7076; Email: sabenner{at}ffame.org
Present address: Makoto Sukeda, Japan Association for International Chemical Information (JAICI), 6-25-4 Honkomagome Bunkyo-ku Tokyo 113-0021, Japan
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