Nucleic Acids Research, Vol 27, Issue 8 1788-1794, Copyright © 1999 by Oxford University Press
K He, KW Porter, A Hasan, JD Briley and and BR Shaw
Direct PCR sequencing with boronated nucleotides provides an alternative to
current PCR sequencing methods. The positions of boranophosphate-modified
nucleotides incorporated randomly into DNA during PCR can be revealed
directly by exonuclease digestion to give sequencing ladders. Cytosine
nucleotides, however, are especially sensitive to exonuclease digestion and
provide suboptimal sequencing ladders. Therefore, a series of 5-substituted
analogs of 2'- deoxycytidine 5'-(alpha-P-borano)triphosphates (dCTPalphaB)
were synthesized with the hope of increasing the nuclease resistance of
deoxycytosine residues and thereby enhancing the deoxycytosine band
intensities. These dCTP analogs contain a boranophosphate modification at
the alpha-phosphate group in 2'-deoxycytidine 5'-triphosphate (dCTP) as
well as a 5-methyl, 5-ethyl, 5-bromo or 5-iodo substitution for the
5-hydrogen of cytosine. The two diastereomers of each new dCTP derivative
were separated by reverse phase HPLC. The first eluted diastereomer
(putatively Rp) of each dCTP analog was a substrate for T7 DNA polymerase
(Sequenase) and had an incorporation efficiency similar to normal dCTP and
dCTPalphaB, with the 5-iodo-dCTPalphaB analog being the least efficient.
Substitution at the C-5 position of cytosine by alkyl groups (ethyl and
methyl) markedly enhanced the dCTPalphaB resistance towards exonuclease III
(5-Et-dCTPalphaB >5-Me-dCTPalphaB >dCTPalphaB approximately
5-Br-dCTPalphaB >5-I-dCTPalphaB), thereby generating DNA sequences that
better define the deoxycytosine positions. The introduction of modified
dCTPalphaB should increase the utility of direct DNA sequencing with
boronated nucleoside 5'- triphosphates.
ARTICLES
Synthesis of 5-substituted 2'-deoxycytidine 5'-(alpha-P- borano)triphosphates, their incorporationinto DNA and effects on exonuclease
Department of Chemistry, P. M. Gross Chemical Laboratory, Duke University, Durham, NC 27708, USA.
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