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

Abstract

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. © The Author 2005. Published by Oxford University Press. All rights reserved.