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

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).