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 ((Me)isoC) 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 d(Me)isoC 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 d(Me)isoC 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 d(Me)isoC-disoG content. Challenges in replication with d(Me)isoC-disoG are consistent with nucleobase tautomerism in the insertion step and disrupted minor groove nucleobase pair-polymerase contacts in subsequent extension.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Ahle JD,Barr S,Chin AM,Battersby TR

doi

10.1093/nar/gki628

keywords:

subject

Has Abstract

pub_date

2005-06-02 00:00:00

pages

3176-84

issue

10

eissn

0305-1048

issn

1362-4962

pii

33/10/3176

journal_volume

33

pub_type

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