Nucleic Acids Research Advance Access originally published online on September 29, 2006
Nucleic Acids Research 2006 34(19):5383-5394; doi:10.1093/nar/gkl637
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Nucleic Acids Research, 2006, Vol. 34, No. 19 5383-5394
© 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.
Molecular Biology |
Systematic characterization of 2'-deoxynucleoside- 5'-triphosphate analogs as substrates for DNA polymerases by polymerase chain reaction and kinetic studies on enzymatic production of modified DNA
1 Department of Applied Chemistry, Faculty of Engineering, Gunma University Gunma 376-8515, Japan 2 PRESTO, Japan Science and Technology Agency (JST) Saitama 332-0012, Japan
*To whom correspondence should be addressed. Tel: +81 277 30 1222; Fax: +81 277 30 1222; Email: kuwahara@chem.gunma-u.ac.jp
Received June 30, 2006. Revised July 28, 2006. Accepted August 14, 2006.
We synthesized C5-modified analogs of 2'-deoxyuridine triphosphate and 2'-deoxycytidine triphosphate and investigated them as substrates for PCRs using Taq, Tth, Vent(exo-), KOD Dash and KOD(exo-) polymerases and pUC 18 plasmid DNA as a template. These assays were performed on two different amplifying regions of pUC18 with different T/C contents that are expected to have relatively high barriers for incorporation of either modified dU or dC. On the basis of 260 different assays (26 modified triphosphates x 5 DNA polymerases x 2 amplifying regions), it appears that generation of the full-length PCR product depends not only on the chemical structures of the substitution and the nature of the polymerase but also on whether the substitution is on dU or dC. Furthermore, the template sequence greatly affected generation of the PCR product, depending on the combination of the DNA polymerase and modified triphosphate. By examining primer extension reactions using primers and templates containing C5-modified dUs, we found that a modified dU at the 3' end of the elongation strand greatly affects the catalytic efficiency of DNA polymerases, whereas a modified dU opposite the elongation site on the template strand has less of an influence on the catalytic efficiency.
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