High-performance liquid chromatographic purification of deoxynucleoside 5'-triphosphates and their use in a sensitive electrophoretic assay of misincorporation during DNA synthesis.
This paper describes techniques and strategies for semi-preparative high-performance liquid chromatographic (HPLC) purification of 2'-deoxynucleoside 5'-triphosphates (dNTPs). The procedure yields dNTPs that are sufficiently pure for use in a sensitive electrophoretic assay of misincorporation during DNA synthesis. Anion-exchange HPLC was used to purify the four normal dNTPs (dATP, dGTP, dCTP and dTTP), plus the chemically modified analogues, 5-BrdUTP, 5-IodUTP and 1,N6-etheno-dATP (epsilon dATP). Baseline separations were achieved by isocratic elution of dNTPs with potassium dihydrogen phosphate mobile phase. In general, the resolution of dNTPs was highly dependent on pH, although the influence of mobile phase composition on separation of dNTPs was not the same for all three HPLC packing materials used. A Hewlett-Packard diode array detector was extremely valuable in the identification of contaminating peaks and in the development of optimal mobile phase conditions for dNTP purification. The pure dNTPs were used in the electrophoretic assay of misincorporation, yielding information about the mispairing potential of the modified dNTPs. BrdUMP and IodUMP were misincorporated in place of dCMP during chain elongation catalyzed by purified DNA polymerase I of Escherichia coli. epsilon dAMP was incorporated into DNA in place of dAMP, although at much lower efficiency than dAMP.
D002848:Chromatography, DEAE-Cellulose / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004247:DNA / Q000096:biosynthesis*; D004256:DNA Polymerase I / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000302:isolation & purification*; D003856:Deoxyuracil Nucleotides / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology; D004985:Ethenoadenosine Triphosphate / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration
D003854:Deoxyribonucleotides; D003856:Deoxyuracil Nucleotides; D004985:Ethenoadenosine Triphosphate; C027078:deoxyuridine triphosphate; C032549:bromodeoxyuridine triphosphate; C008144:5-iodo-2'-deoxyuridine triphosphate; D004247:DNA; D004256:DNA Polymerase I