Effect of transition metal ions on the fluorescence and Taq-catalyzed polymerase chain reaction of tricyclic cytidine analogs

The cytosine analogs 1,3-diaza-2-oxophenothiazine (tC) and 1,3-diaza-2-oxophenoxazine (tCo) stand out among fluorescent bases due to their unquenched fluorescence emission in double-stranded DNA. Recently, we reported a method for the generation of densely tCo-labeled DNA by polymerase chain reaction (PCR) that relied on the use of the extremely thermostable Deep Vent polymerase. We have now developed a protocol that employs the more commonly used Taq polymerase. Supplementing the PCR with Mn2+ or Co2+ ions dramatically increased the amount of tCo triphosphate (dtCoTP) incorporated and, thus, enhanced the brightness of the PCR products. The resulting PCR products could be easily detected in gels based on their intrinsic fluorescence. The Mn2+ ions modulate the PCR by improving the bypass of template tCo and the overall catalytic efficiency. In contrast to the lower fidelity during tCo bypass, Mn2+ improved the ability of Taq polymerase to distinguish between dtCoTP and dTTP when copying a template dA. Interestingly, Mn2+ ions hardly affect the fluorescence emission of tC(o), whereas the coordination of Co2+ ions with the phosphate groups of DNA and nucleotides statically quenches tC(o) fluorescence with small reciprocal Stern–Vollmer constants of 10–300 μM.