The Efficiency of DNA Labeling with Near-Infrared Fluorescent Dyes

摘要

The efficiency of DNA labeling was assessed for 2'-deoxyuridine 5'-triphosphate (dUTP) derivatives containing the Cy7 cyanine dye as a fluorophore. Two fluorescent Cy7-labeled dUTP analogs differed in the chemical structure of the linker between the fluorophore and nucleotide moieties. The efficiency of the polymerase chain reaction (PCR) and inhibition with modified nucleotides were estimated by real-time PCR. The efficiency of labeled nucleotide incorporation in PCR products was measured by quantitative electrophoresis. The efficiency of target DNA labeling was evaluated by binding the fluorescently labeled PCR products to a microarray of oligonucleotide probes immobilized in hydrogel drops (a biochip). The near-infrared hybridization signal was detected by digital luminescence microscopy. An increase in linker length was found to provide more efficient incorporation of the labeled nucleotide. Both of the compounds provided high sensitivity and high specificity of DNA testing via allele-specific hybridization on a biochip.