Heterodimeric DNA-binding dyes designed for energy transfer: stability and applications of the DNA complexes

Abstract

Spectroscopic studies of the complexes of double-stranded (ds) DNA with the polymethylene-amine linked heterodimers thiazole orange-thiazole blue, thiazole orange-ethidium, and fluorescein-ethidium, in each case show efficient energy transfer from donor to acceptor chromophores (Benson, S.C., Singh, P. and Glazer, A.N. (1993) accompanying manuscript). A quantitative assay of the stability of such complexes during gel electrophoresis is presented. The off-rate of dye from complexes formed at an initial dsDNA bp:dye ratio > or = 10:1 follows strict first-order kinetics. The t0.5 values for the dissociation of a series of related dyes provide a quantitative criterion for the design of DNA-binding fluorophores. Complexes of dsDNA with the monomeric propidium and cyanine dyes, [1-(9-amino-4,7-diazanonyl)-3,8-diamino-6-phenyl-phenanthridinium bromide trihydrobromide] and (N,N'-tetramethyl-1,3-propanediamino)propyl thiazole orange [4-[3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidenyl]-1-(4 ,4,8-trimethyl-4,8-diazanonyl)-quinolinium diiodide], are much more stable than those with their widely used counterparts, ethidium and thiazole orange. Applications of the new dyes in post-staining of gels and in the multiplex detection of DNA restriction fragments are presented.

Rapid detection of low-abundance K-ras mutation in stools of colorectal cancer patients using chip-based temperature gradient capillary electrophoresis

“…Meanwhile, unconjugated EtBr itself will cause a high-fluorescence background and interferes with the detection sensitivity. 23,44,45 Although our preliminary clinical investigation is limited because of restricted access to more CRC samples, the results of our study showed that the detection accuracy rate reached 100% after verification by clone sequencing. The high accuracy might be due to the relatively high sensitivity of this method, because the mutations harbored in stools can be as little as 1% in general, 11 which is much higher than the threshold concentration (0.2%) of this chip-based TGCE method.…”

“…YOYO-1 is essentially nonfluorescent in the absence of nucleic acids and exhibits a 100-to 1000-fold increase in fluorescence upon binding to DNA; therefore, this system can detect as low as 0.5 ng/ml of double-stranded DNA (dsDNA). 44,45 In contrast, the fluorescence of the commonly used EtBr is only enhanced B20-to 30-fold after intercalation into dsDNA, which requires a relatively high concentration of DNA sample to be effective in the TGCE assay (50-500 ng/ml). Meanwhile, unconjugated EtBr itself will cause a high-fluorescence background and interferes with the detection sensitivity.…”

Microwave synthesis and fluorescence properties of homo- and heterodimeric monomethine cyanine dyes TOTO and their precursors

“…Interest in nonradioactive DNA stains that are stable under gel electrophoretic conditions has led to the synthesis and characterization of a family of homo-and heterodimeric DNA-binding dyes (1)(2)(3). Organic fluorescent probes (FPs) are, as a rule, polycyclic aromatic cations with the planar structure capable of incorporating (intercalating) between the planes of the DNA bases, pulling apart these planes and changing the structure of nucleic acids.…”

“…Bis-intercalating TOTO-1 dyes have a weak fluorescence in the free state, but they sharply (more than 1000 times) increase fluorescence due to binding with DNA (4) and, therefore, they are widely used in biological, medical and drug development areas as fluorescent labels and probes (8)(9)(10)(11)(12)(13)(14)(15). They found also applications in genetic studies and modern diagnostic methods (1)(2)(3), for examples, in the case of the polymerase chain reaction for cancer diagnostics at early stages of the disease; for diagnostics of infection diseases (1)(2)(3)(4), including AIDS; for the identification of DNA samples in criminal law (16,17); flow cytometry (18), DNA sequencing (19,20) and quantification of nucleic acids in capillary and gel electrophoresis (21)(22)(23). Besides, they are commonly applied to lasers (24), electronics (25), nonlinear optics (26) and solar cells (27).…”

Prestaining Method as a Useful Tool for the Agarose Gel Electrophoretic Detection of Polymerase Chain Reaction Products with a Fluorescent Dye SYBR® Gold Nucleic Acid Gel Stain

“…2 The prestain method has some advantages in that it can be practiced with smaller amounts of staining dyes, and does not need any time-consuming poststain process. However, the method has some disadvantages, in that the mobility of DNA-dye complexes is affected by some factors, such as the dye:DNA concentration ratio [3][4][5][6] and the dye-DNA base pair (bp) ratio. [5][6][7] A caution has been reported by a SYBR Gold supplier, 2 i.e.…”