Interaction of ROX and ssGreen Fluorophores Affects Visualization of Gel Electrophoresis Results

Abstract

Fluorescent molecules have found wide application in biological and medical research: from flow cytometry and confocal microscopy to ELISA and PCR methods. Understanding the properties of fluorophores and their interactions is an integral part of developing new research methods in a wide range of scientific fields. In this paper, we present the results of the analysis of the interaction of carboxy-X-rhodamine (ROX) and the intercalating dye of the green spectrum for single-stranded DNA and RNA molecules – ssGreen. It was found that the presence of ssGreen in the agarose gel visually gives a faint enhance to the rhodamine signal compared to the gel without ssGreen. Moreover, the absence of green spectrum luminescence (quenching) was observed in the oligonucleotide with rhodamine in its composition, in contrast to the PCR primer without fluorophores in its composition. The proposed FRET phenomenon is not supported by the results of quantum mechanism calculations, which requires further study.