High-sensitivity two-color detection of double-stranded DNA with a confocal fluorescence gel scanner using ethidium homodimer and thiazole orange (original) (raw)
Related papers
Nucleic Acids Research, 1992
The synthesis, proof of structure, and the absorption and fluorescence properties of two new unsymmetrical cyanine dyes, thiazole orange dimer (TOTO; 1,1 '-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methyl-2,3-dihydro-(benzo-1 ,3-thiazole)-2-methylidene]-quinolinium tetraiodide) and oxazole yellow dimer (YOYO; an analogue of TOTO with a benzo-1,3oxazole in place of the benzo-1,3-thiazole) are reported. TOTO and YOYO are virtually non-fluorescent in solution, but form highly fluorescent complexes with double-stranded DNA (dsDNA), up to a maximum dye to DNA bp ratio of 1:4, with >1000-fold fluorescence enhancement. The dsDNA-TOTO (Xmax 513 nm; miax 532 nm) and dsDNA-YOYO (Xmax 489 nm; Xax 509 nm) complexes are completely stable to electrophoresis on agarose and acrylamide gels. Mixtures of restriction fragments pre-labeled with ethidium dimer (EthD; max 616 nm) and those pre-labeled with either TOTO or YOYO were separated by electrophoresis. Laser excitation at 488 nm and simultaneous confocal fluorescence detection at 620-750 nm (dsDNA-EthD emission) and 500-565 nm (dsDNA-TOTO or dsDNA-YOYO emission) allowed sensitive detection, quantitation, and accurate sizing of restriction fragments ranging from 600 to 24,000 bp. The limit of detection of dsDNA-TOTO and YOYO complexes with a laserexcited confocal fluorescence gel scanner for a band 5-mm wide on a 1-mm thick agarose gel was 4 picograms, about 500-fold lower than attainable by conventional staining with ethidium bromide.
Photochemistry and Photobiology, 2001
The absorption and fluorescence spectra, fluorescence quantum yields, lifetimes and time-resolved fluorescence spectra are reported for nine different fluorescent DNAdyes. The work was initiated in search of a quantitative method to detect the ratio of single-to-double stranded DNA (ssDNA/dsDNA) in solution based on the photophysics of dye-DNA complexes; the result is a comprehensive study providing a vast amount of information for users of DNA stains. The dyes examined were the bisbenzimide or indole-derived stains (Hoechst 33342, Hoechst 33258 and 4,6-diamidino-2-phenylindole), phenanthridinium stains (ethidium bromide and propidium iodide) and cyanine dyes (PicoGreen, YOYO-1 iodide, SYBR Green I and SYBR Gold). All were evaluated under the same experimental conditions in terms of ionic strength, pH and dye-DNA ratio. Among the photophysical properties evaluated only fluorescence lifetimes for the cyanine stilbene dyes allowed a convenient differentiation between ssDNA and dsDNA. The bisbenzimide dyes showed multiexponential decays when bound to either form of DNA, making lifetime-based analysis cumbersome with inherent errors. These dyes also presented biexponential decay when free in aqueous buffered solutions at different pH. A mechanism for their deactivation is proposed based on two different conformers decaying with different kinetics. The phenanthridinium dyes showed monoexponential decays with ssDNA and dsDNA, but there was no discrimination between them. High dye-DNA ratios (e.g. 1:1) resulted in multiexponential decays for cyanine dyes, resulting from energy transfer or self-quenching deactivation. Shifts in both absorption and fluorescence maxima for both ssDNA and ds-DNA DNA-cyanine dye complexes were small. Broadening of dye-ssDNA absorption and fluorescence bands for the cyanine dyes relative to dye-dsDNA bands was detected and attributed to higher degrees of rotational freedom in the former. ¶Posted on the website on 23 March 2001.
Analytical Biochemistry, 1977
Ultraviolet sources with output wavelengths of 254,300, and 366 nm were compared for detection of ethidium bromide-DNA complexes in acrylamide gels. The 254-and 300-nm sources were both much more sensitive than the 366-nm source. The 254-nm source produced a great deal of photodamage, photonicking and photodimerization, and photobleaching, while the longer wavelength sources cause little damage or bleaching. The 300-nm source is clearly the most suitable source, providing high sensitivity and a relatively low amount of photodamage and photobleaching. The examination of DNA on polyacrylamide or agarose gels using fluorescent staining with ethidium bromide (EB) is convenient and effective (1). Ultraviolet sources for exciting the EB-DNA complex are commercially available, with output at either 254 or 366 nm. Each of these sources has its shortcomings. The 254-nm source produces photodamage in the nucleic acid and causes photobleaching of the complex. The 366nm source is relatively inefficient and gives a weak fluorescence of the complex. The excitation maximum of an EB-DNA complex occurs in the range of 300 nm, and fluorescent lamps that emit in this range are commercially available. We have constructed an ultraviolet source using these 300-nm lamps in conjunction with a filter that passes the ultraviolet and blocks visible wavelengths. This system should be optimal for the fluorescent detection of EB-DNA complexes. In this paper, we compare the 300-nm source with commercially available 254-and 366-nm sources as a means of detecting EB-stained DNA bands on an acrylamide gel. METHODS Construction of a 300-nm source. The 300-nm source was constructed using eight 8-W 300-nm fluorescent lamps (RPR-3000 A).l These lamps
Concerted Intercalation and Minor Groove Recognition of DNA by a Homodimeric Thiazole Orange Dye
Bioconjugate Chemistry, 2000
The thiazole orange dye TOTO binds to double-stranded DNA (dsDNA) by a sequence selective bisintercalation. Each chromophore is sandwiched between two base pairs in a (5′-CpT-3′):(5′-ApG-3′) site, and the linker spans two base pairs in the minor groove. We have used one-and two-dimensional NMR spectroscopy to examine the dsDNA binding of an analogue of TOTO in which the linker has been modified to contain a bipyridyl group (viologen) that has minor groove binding properties. We have investigated the binding of this analogue, called TOTOBIPY, to three different dsDNA sequences containing a 5′-CTAG-3′, a 5′-CTTAG-3′, and a 5′-CTATAG-3′ sites, respectively, demonstrating that TOTOBIPY prefers to span three base pairs. The many intermolecular NOE connectivities between TOTOBIPY and the d(CGCTTAGCG):d(CGCTAAGCG) oligonucleotide in the complex shows that the bipyridyl-containing linker is positioned in the minor groove and spans three base pairs. Consequently, we have succeeded in designing and synthesizing a ligand that recognizes an extended recognition sequence of dsDNA as the result of a concerted intercalation and minor groove binding mode.
Bioconjugate Chemistry, 2008
We have designed a doubly thiazole orange labeled nucleoside showing high fluorescence intensity for a hybrid with the target DNA and effective quenching for a single-stranded state. Knowing how much the fluorescence emission and quenching of this probe depend on the probe sequence and why there is such a sequence dependence is important for effective probe design, we synthesized more than 30 probe sequences and measured their fluorescence intensities. When the probe hybridized with the target DNA strands, there was strong emission, whereas the emission intensity was much weaker before hybridization; however, self-dimerization of probes suppressed fluorescence quenching. In particular, the G/C base pairs neighboring the labeled nucleotide in a self-dimeric structure resulted in a low quenching ability for the probe before hybridization. On the other hand, mismatched base pair formation around the labeled site decreased the fluorescence intensity because the neighboring sequence is the binding site of the tethered thiazole orange dyes. The hybridization enhanced the fluorescence of the probe even when the labeled nucleotide was located at the end of the probe strand; however, the partial lack of duplex structure resulted in a decrease in the fluorescence intensity of the hybrid.
Fluorescence of Styryl Dyes-DNA Complexes Induced by Single and Two-Photon Excitation
Journal of Fluorescence, 2006
The series of novel monomer and homodimer styryl dyes based on (p-dimethylaminostyryl) benzothiazolium residues were synthesized and studied as possible fluorescent probes for nucleic acids detection. Spectral-luminescent and spectral-photometric properties of obtained dyes in the unbound state and in DNA presence were studied. Fluorescence emission induced by two-photon excitation of dye-DNA complexes in aqueous buffer solution was registered. Two-photon absorption cross section values of the studied dyes in DNA presence were evaluated.