Amplification Efficiency and Substrate Properties of Fluorescently Labeled Deoxyuridine Triphosphates in PCR in the Presence of DNA Polymerases without 3'-5' Exonuclease Activity

Abstract

The kinetics of PCR amplification has been studied using fluorescently labeled zwitterionic deoxyuridine triphosphates that contain different fluorophores linkers of different lengths between the fluorophore and nitrogenous base. The concentration dependence of the modified substrate on the yield of the PCR product has been studied. Taq and Vent (exo–) DNA polymerases without 3'-5' exonuclease activity have been used for amplification, and the subsequent hybridization analysis of amplification products has been performed. Hydrophobicity and spatial size of the fluorophore have been found to affect inhibition of polymerases in PCR, while the length of the linker between the fluorophore and heterocyclic base has a significant impact on the substrate properties of the labeled deoxyuridine derivatives, i.e., on their incorporation in the PCR product. Electroneutral derivatives of deoxyuridine triphosphate with an average linker length have appeared to be most effective in PCR and subsequent microarray hybridization analysis. The studied fluor-dUTP are suitable for the incorporation of labels in PCR products using Taq and Vent (exo–) DNA polymerases in the subsequent hybridization analysis.