ELIMINATION OF RESIDUAL NATURAL NUCLEOTIDES FROM 3'-O-MODIFIED-DNTP SYNTHESES BY ENZYMATIC MOP-UP

Citation
Ml. Metzker et al., ELIMINATION OF RESIDUAL NATURAL NUCLEOTIDES FROM 3'-O-MODIFIED-DNTP SYNTHESES BY ENZYMATIC MOP-UP, BioTechniques, 25(5), 1998, pp. 814-817
Citations number
3
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biology,"Biochemical Research Methods
Journal title
BioTechniques → ACNP
ISSN journal
0736-6205
Volume
25
Issue
5
Year of publication
1998
Pages
814 - 817
Database
ISI
SICI code
0736-6205(1998)25:5<814:EORNNF>2.0.ZU;2-Q
Abstract
Here, Mle describe a novel strategy called enzymatic ''Mop-Up'' that e fficiently removes contaminating dNTPs from reverse-phase, high-perfor mance liquid chromatography (RP-HPLC) purified 3'-O-modified dNTP synt heses. Enzymatic mop-up, takes advantage of the high selectivity of DN A polymerases for the former nucleoside triphosphates over the latter nucleotide analogs. We demonstrate the selective removal of contaminat ing dATP and dTTP from RP-HPLC purified 3'-O-methyl-dATP and 3'-O-(2-n itrobenzyl)-dTTP syntheses, respectively. These data highlight the imp ortance of natural nucleotide contamination when interpreting enzymati c incorporation data and provide an alternative hypothesis for the obs erved property of catalytic editing of DNA polymerases. Moreover, the effective removal of natural nucleotides from 3'-O-modified analogs ad dresses the important issue of nucleotide read-through for stop-start DNA sequencing strategies, such as the bose addition sequencing scheme (BASS).