Tricolour fluorescence detection of sequence-specific DNA with a new molecular beacon and a nucleic acid dye TOTO-3

Abstract

We have developed a tricolor fluorescence quantitative method for sequence-specific DNA detection using a new molecular beacon (MB) and a nucleic acid dye TOTO-3. This new MB is designed with two fluorophores of FAM and TAMRA instead of one fluorophore and one quencher of traditional MB, and a nucleotide with guanine base is attached directly to FAM as a quencher. In the absence of target DNA, MBs are in the stem–loop state. The fluorescence of FAM is absorbed by TAMRA, and the fluorescence of TAMRA is quenched by guanine base. Meanwhile, the interaction between TOTO-3 and MBs is very weak. In the presence of target DNA, MBs hybridize with target DNA to form a double-stranded structure. TAMRA is separated from FAM and guanine base, and the fluorescence of FAM and TAMRA recovers simultaneously. At the same time TOTO-3 binds to double-stranded DNA, the fluorescence of TOTO-3 significantly enhances. In this strategy, the false-positive signals of MBs caused by non-specific interactions can be distinguished by the change of the ratio of the total fluorescence intensities of FAM and TAMRA to that of TOTO-3 at different concentrations of target DNA. In the simple sample, the detection of target DNA can be achieved with the total fluorescence intensity of three dyes, which results in a significant improvement of the detection sensitivity. In the complex sample, the detection of target DNA can be achieved with the fluorescence intensity of TOTO-3 which can overcome the false-positive signals of MBs and improve the detection accuracy.