Journal: Nucleic Acids Research

Article Title: Highly efficient incorporation of the fluorescent nucleotide analogs tC and tC O by Klenow fragment

doi: 10.1093/nar/gkp266

Figure Lengend Snippet: Chemical structure and base pairing pattern of the fluorescent base analogs tC and tC O . Absorption and emission spectra of the free monomers of tC (dashed line) and tC O (solid line). The fluorescence emission is normalized to represent the relative quantum yields.

Article Snippet: The bands were quantified using a Typhoon fluorescence scanner (Molecular Dynamics).

Techniques: Fluorescence

Journal: Nucleic Acids Research

Article Title: Highly efficient incorporation of the fluorescent nucleotide analogs tC and tC O by Klenow fragment

doi: 10.1093/nar/gkp266

Figure Lengend Snippet: Formation of the ternary KF–DNA–dNTP complex probed by steady-state fluorescence anisotropy. ( a ) In three separate experiments, 125 nM (black triangles), 250 nM (gray circles) and 500 nM (black squares) dtCTP was incubated with increasing amounts of KF in the presence of 10 μM primer/template (X = G). In all binding experiments, the 3′-end of the primer was terminated with a 2′,3′-dideoxy-cytosine. ( b ) The same experiments were carried out using 75 nM (black triangles), 125 nM (gray circles) and 250 nM (black squares) dtC O TP. The affinity curves resulting from the best fits (see text) are shown as solid curves.

Article Snippet: The bands were quantified using a Typhoon fluorescence scanner (Molecular Dynamics).

Techniques: Fluorescence, Incubation, Binding Assay

Journal: Nucleic Acids Research

Article Title: Highly efficient incorporation of the fluorescent nucleotide analogs tC and tC O by Klenow fragment

doi: 10.1093/nar/gkp266

Figure Lengend Snippet: Competitive binding curves. The samples contained 10 µM primer/template (X = G), 203 nM KF and 250 nM dtCTP ( a ) or 250 nM dtC O TP ( b ). The stepwise addition of dCTP (black squares) replaces the fluorescent nucleoside triphosphates in the ternary complexes. Insets: Effect of protein binding on the fluorescence emission of dtCTP (inset in a) and dtC O TP (inset in b). Total 125 nM nucleotide analog were incubated with 284 nM Klenow fragment and 10 µM primer/template (X = G) until the fluorescence remained stable. Spectra of the free nucleotide analogs are shown as black lines and spectra of the nucleotides in complex with KF are shown as black dots.

Article Snippet: The bands were quantified using a Typhoon fluorescence scanner (Molecular Dynamics).

Techniques: Binding Assay, Protein Binding, Fluorescence, Incubation