Nucleic Acids Research Advance Access first published online on August 31, 2006
This version published online on September 8, 2006
Nucleic Acids Research, doi:10.1093/nar/gkl632
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© 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.
Methods Online |
A mass spectrometry-based approach for identifying novel DNA polymerase substrates from a pool of dNTP analogues
Department of Chemistry and Biochemistry, University of Colorado Boulder, CO 80309, USA
*To whom correspondence should be addressed. Tel: +1 303 492 7027; Fax: +1 303 492 5894; Email: Kuchta{at}colorado.edu
Received May 11, 2006. Revised July 24, 2006. Accepted August 10, 2006.
There has been a long-standing interest in the discovery of unnatural nucleotides that can be incorporated into DNA by polymerases. However, it is difficult to predict which nucleotide analogs will prove to have biological relevance. Therefore, we have developed a new screening method to identify novel substrates for DNA polymerases. This technique uses the polymerase itself to select a dNTP from a pool of potential substrates via incorporation onto a short oligonucleotide. The unnatural nucleotide(s) is then identified by high-resolution mass spectrometry. By using a DNA polymerase as a selection tool, only the biologically relevant members of a small nucleotide library can be quickly determined. We have demonstrated that this method can be used to discover unnatural base pairs in DNA with a detection threshold of 
10% incorporation.