A Novel Fluorescent GFP Chromophore Analog-Based Dye for Quantitative PCR

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A. A. Stakheev^1,a*, D. Yu. Ryazantsev¹, Yu. K. Zvezdina¹, M. S. Baranov¹, and S. K. Zavriev¹

¹Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia

^* To whom correspondence should be addressed.

Received February 20, 2018; Revision received April 20, 2018

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This is the first report describing the possibility of using a green fluorescent protein chromophore synthetic analog, P-HOBDI-BF₂, as a fluorescent dye for a linear hydrolysis probe used in qPCR. The study was carried out on a system for detection of the plant pathogenic fungus Fusarium avenaceum using a plasmid containing translation elongation factor 1α fragment as a template. To estimate fluorogenic properties of P-HOBDI-BF₂, 6-FAM- and BDP-FL-labeled probes were used. It was demonstrated that a synthetic dye based on the P-HOBDI-BF₂ chromophore can be used for labeling hydrolysis probes for qPCR, but fluorescence increase levels for P-HOBDI-BF₂-labeled probes were slightly lower than those for 6-FAM-labeled ones. At the same time, the sensitivity of P-HOBDI-BF₂-based assays remained high, and this fact together with acceptable fluorescence levels suggests that this dye can be considered as an efficient alternative for reporters traditionally used for fluorescence detection in the FAM channel.

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KEY WORDS: quantitative PCR, fluorophore, probe, green fluorescent protein, fluorescence increase

DOI: 10.1134/S000629791807009X

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