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Nucleic Acids Research Advance Access originally published online on December 10, 2008
Nucleic Acids Research 2009 37(2):e14; doi:10.1093/nar/gkn956
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Nucleic Acids Research, 2009, Vol. 37, No. 2 e14
© 2008 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.

Methods Online

An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules

Michiko Kimoto1,2, Rie Kawai1, Tsuneo Mitsui2, Shigeyuki Yokoyama1,3 and Ichiro Hirao1,2,*

1Systems and Structural Biology Center, RIKEN, 2TagCyx Biotechnologies, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045 and 3Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

*To whom correspondence should be addressed. Tel: +81 45 503 9644; Fax: +81 45 503 9645; Email: ihirao{at}riken.jp

Received October 6, 2008. Revised November 5, 2008. Accepted November 11, 2008.

Toward the expansion of the genetic alphabet, we present an unnatural base pair system for efficient PCR amplification, enabling the site-specific incorporation of extra functional components into DNA. This system can be applied to conventional PCR protocols employing DNA templates containing unnatural bases, natural and unnatural base triphosphates, and a 3'->5' exonuclease-proficient DNA polymerase. For highly faithful and efficient PCR amplification involving the unnatural base pairing, we identified the natural-base sequences surrounding the unnatural bases in DNA templates by an in vitro selection technique, using a DNA library containing the unnatural base. The system facilitates the site-specific incorporation of a variety of modified unnatural bases, linked with functional groups of interest, into amplified DNA. DNA fragments (0.15 amol) containing the unnatural base pair can be amplified 107-fold by 30 cycles of PCR, with <1% total mutation rate of the unnatural base pair site. Using the system, we demonstrated efficient PCR amplification and functionalization of DNA fragments for the extremely sensitive detection of zeptomol-scale target DNA molecules from mixtures with excess amounts (pmol scale) of foreign DNA species. This unnatural base pair system will be applicable to a wide range of DNA/RNA-based technologies.


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G. T. Hwang, Y. Hari, and F. E. Romesberg
The effects of unnatural base pairs and mispairs on DNA duplex stability and solvation
Nucleic Acids Res., August 1, 2009; 37(14): 4757 - 4763.
[Abstract] [Full Text] [PDF]


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