Simultaneous Incorporation of Different Cy5-Labeled Deoxypyrimidine Nucleotides into the Synthesized DNA Chain

In PCR with Taq polymerase on the Staphylococcus aureus genomic DNA template, the substrate properties of eight fluorescently labeled deoxyuridine and deoxycytidine triphosphates (Cy5-dUTP and Cy5-dCTP), which are dU-dC pairs with similar cyanine substituents, was compared during simultaneous introduction of the such pairs in PCR. The different Cy5-dUTP and Cy5-dCTP pairs differed from each other in that each of them had substituents with different linker lengths between the nitrogenous base and the fluorophore, as well as the linker lengths between the quaternary ammonium group and the second heterocycle of the Cy5 fluorophore. The amplification efficiency, as well as the yield of the product, and the density of label incorporation were determined. It was found that with the simultaneous introduction of Cy5-modified dU and dC into the reaction at equimolar concentrations, the inhibitory effect was not directly proportional to the concentration, in contrast to that with separate (individual) introduction of fluorescently labeled dNTPs. This allows one to use the simultaneous introduction of Cy5-modified dU and dC into PCR to increase sensitivity in methods based on the detection of a fluorescent signal, for example, in DNA-microarray technology.

fluorescently labeled nucleoside triphosphates, substrate efficiency, PCR, kinetics