Fluorescence Labeling of GC-Rich DNA Matrix with Different Nucleotide Derivatives for Hybridization Analysis on a Biological Microarray

Objective: We investigated the efficiency of labeling the GC-rich promoter region of the TERT gene in the human genome with derivatives of 5′-triphosphates of 2′-deoxyuridine (dU) and 2'-deoxycytidine (dC) containing cyanine dyes Cy5 and Cy7 as a fluorescent label. Methods: The effect of modified nucleotides on the efficiency of the polymerase chain reaction was evaluated by real-time PCR, and the extent of nucleotide incorporation into the PCR product was also determined. The efficiency of DNA matrix labeling was determined by the intensity of fluorescent signal during allele-specific hybridization on a biological microarray (biochip). Results and Discussion: The highest level of biochip gel drops fluorescence was observed for dU-Cy7 derivatives compared to dU- and dC-Cy5 derivatives. At the same time, in the case of GC-rich DNA matrix, the use of dC-Cy5 derivatives provided a fundamentally better result compared to dU-Cy5 derivative. Thus, modified Cy7 analogs capable of incorporation into DNA during PCR are less dependent on the GC composition of the DNA matrix and are more universal fluorescent labels for diagnostic purposes. Conclusions: Further application of modified Cy7 analogs in the development of laboratory-on-a-chip test systems seems to be the most promising.