title: 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.
authors.affiliation: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, 430072, China.
authors.forename: Dongshan
authors.initials: D
authors.lastname: Xiang
authors.forename: Cuiling
authors.initials: C
authors.lastname: Zhang
authors.forename: Lu
authors.initials: L
authors.lastname: Chen
authors.forename: Xinghu
authors.initials: X
authors.lastname: Ji
authors.forename: Zhike
authors.initials: Z
authors.lastname: He
mesh_terms: D001483:Base Sequence; D002021:Buffers; D003116:Color; D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000235:genetics*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D057230:Limit of Detection; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000737:chemistry* / Q000235:genetics; D009994:Osmolar Concentration; D020641:Polymorphism, Single Nucleotide; D011804:Quinolines / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013696:Temperature; D013844:Thiazoles / Q000737:chemistry*
chemical_list: D002021:Buffers; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D011804:Quinolines; C495966:TOTO-3 compound; D013844:Thiazoles; D004247:DNA