The Effect of the Structure of Fluorescently Labeled Nucleotide Derivatives on the Efficiency of Their Incorporation in DNA in the Polymerase Chain Reaction

The efficiency of fluorescence DNA labeling was estimated for four fluorescent 2′-deoxyuridine 5′-triphosphate derivatives differing in the orientation of the main dye axis, which passes through the polymethine chain, relative to the linker connecting the dye to the nucleotide. To estimate the polymerase chain reaction (PCR) rate, real-time PCR was run with two commercial hot-start DNA polymerases possessing 5′→3′ exonuclease activity in the presence of an intercalating dye. The efficiency of the test compound incorporation in the PCR product was estimated via a quantitative analysis of the amplification product by agarose gel electrophoresis. The fluorescently labeled product was then hybridized on a biological microchip and the ratio of signals from perfect match and mismatch duplexes was determined. The incorporation efficiency and discrimination between perfect match and mismatch duplexes were found to depend on the relative orientation of the dye and the linker between the dye and pyrimidine base, as well as on the presence of hydrophilic groups in the dye. Compounds that are efficiently incorporated in a growing DNA strand and show a high specificity in hybridization analysis were identified using biochips.