Elimination of residual natural nucleotides from 3'-O-modified-dNTP syntheses by enzymatic mop-up.
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).
Metzker
M L
ML
Baylor College of Medicine, Houston, TX, USA.
Raghavachari
R
R
Burgess
K
K
Gibbs
R A
RA
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000096:biosynthesis*; D004259:DNA-Directed DNA Polymerase; D003854:Deoxyribonucleotides / Q000138:chemical synthesis / Q000302:isolation & purification*
D003854:Deoxyribonucleotides; D004247:DNA; D004259:DNA-Directed DNA Polymerase