Preparation of Radioisotope-Labeled DNA Substrates

[³²P]ATP was purchased from Perkin Elmer at 6000 Ci/mmol. T₄ polynucleotide kinase and the Klenow fragment of E. coli DNA polymerase I with the exonuclease activity inactivated (KF⁻) were obtained from US Biological (Salem, MA). The dNTPs (ultrapure grade) were purchased from Amersham Biosciences, 5′-(α-P-thio)triphosphates were purchased from TriLink Biotechnologies (San Diego, CA), and the concentrations were determined by UV absorbance.⁴⁴ 1-[(1R, 3S,4R)-3-hydroxy-4-(hydroxymethyl)cyclopentyl]-2,4(1H,3H)-pyrimidinedione (dcUTP) was synthesized as described.^{42 (link)} The phosphoramidites for O⁶mG, and oxoG were purchased from Glen Research (Sterling, VA). The oligodeoxynucleotides containing 6-amino-1-(2-deoxy-β-D-erythro-pentofuranosyl)-1,5-dihydro-4H-imidazo[4,5-c]pyridine-4-one (3DG), O²-POB-T, and N²-BP-dG were synthesized as described.^{45 (link)–47 (link)} The sequence of the template strand was 5′-CTG CGA CAX CTG CGT CTG CGG TGC-3′ with X being A,C,G,T, O⁶mG, oxoG, N²-BP-G, and O²-POB-T. The primer strand was 5′-GCA CCG CAG ACX-3′ with X being G or 3DG. The primer strands were ³²P-labeled with γ-[³²P]ATP in a reaction catalyzed by T₄-polynucleotide kinase, the oligomer was separated from low molecular weight impurities with a spin column containing Sephadex G-25 and the primers were annealed with a 25% excess of the template strands.^{48 (link)}