Elimination of residual natural nucleotides from 3'-O-modified-dNTP syntheses by enzymatic mop-up.

 
TitleElimination of residual natural nucleotides from 3'-O-modified-dNTP syntheses by enzymatic mop-up.
Publication TypeJournal Article
Year of Publication1998
AuthorsMetzker, ML, Raghavachari, R, Burgess, K, Gibbs, RA
JournalBiotechniques
Volume25
Issue5
Pagination814-7
Date Published1998 Nov
ISSN0736-6205
KeywordsBase Sequence, Chromatography, High Pressure Liquid, Deoxyribonucleotides, DNA, DNA-Directed DNA Polymerase
Abstract

Here, we describe a novel strategy called enzymatic "Mop-Up" that efficiently removes contaminating dNTPs from reverse-phase, high-performance liquid chromatography (RP-HPLC) purified 3'-O-modified dNTP syntheses. Enzymatic mop-up takes advantage of the high selectivity of DNA polymerases for the former nucleoside triphosphates over the latter nucleotide analogs. We demonstrate the selective removal of contaminating dATP and dTTP from RP-HPLC purified 3'-O-methyl-dATP and 3'-O-(2-nitrobenzyl)-dTTP syntheses, respectively. These data highlight the importance of natural nucleotide contamination when interpreting enzymatic incorporation data and provide an alternative hypothesis for the observed property of catalytic editing of DNA polymerases. Moreover, the effective removal of natural nucleotides from 3'-O-modified analogs addresses the important issue of nucleotide read-through for stop-start DNA sequencing strategies, such as the base addition sequencing scheme (BASS).

DOI10.2144/98255st01
Alternate JournalBioTechniques
PubMed ID9821582
Grant List1 R01 AI33334 / AI / NIAID NIH HHS / United States
1 R01 HG01459 / HG / NHGRI NIH HHS / United States
HG/GM01745 / HG / NHGRI NIH HHS / United States