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Nucleic Acids Research 2005 33(10):3176-3184; doi:10.1093/nar/gki628
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Published online 2 June 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org

Article

Sequence determination of nucleic acids containing 5-methylisocytosine and isoguanine: identification and insight into polymerase replication of the non-natural nucleobases

J. David Ahle, Stephen Barr, A. Michael Chin1 and Thomas R. Battersby*

Bayer HealthCare LLC, Diagnostics Division 725 Potter Street, Berkeley, CA 94710, USA 1Sequetech Corporation 935 Sierra Vista Avenue, Mountain View, CA 94043, USA

*To whom correspondence should be addressed at PO Box 2466, Berkeley, CA 94702, USA. Tel: +1 510 705 5979; Fax: +1 510 705 5938; Email: thomas.battersby.b{at}bayer.com

Received January 12, 2005. Revised April 1, 2005. Accepted May 13, 2005.

Nucleobase analogs 5-methylisocytosine (MeisoC) and isoguanine (isoG) form a non-natural base pair in duplex nucleic acids with base pairing specificity orthogonal to the natural nucleobase pairs. Sequencing reactions were conducted with oligodeoxyribonucleotides (ODNs) containing dMeisoC and disoG using modified pyrosequencing and dye terminator methods. Modified dye terminator sequencing was generally useful for the sequence identification of ODNs containing the non-natural nucleobases. The two sequencing methods were also used to monitor nucleotide incorporation and subsequent extension by Family A polymerases used in the sequencing methods with a six-nucleobase system that includes dMeisoC and disoG. Nucleic acids containing the six-nucleobase system could be replicated well, but not as well as natural nucleic acids, especially in regions of high dMeisoC–disoG content. Challenges in replication with dMeisoC–disoG are consistent with nucleobase tautomerism in the insertion step and disrupted minor groove nucleobase pair–polymerase contacts in subsequent extension.

This work is dedicated to the memory of our colleague and dear friend Steve Barr, who died on July 18, 2004


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