Published online 2 June 2005
Article |
Sequence determination of nucleic acids containing 5-methylisocytosine and isoguanine: identification and insight into polymerase replication of the non-natural nucleobases
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