yo-pro-1 and thiazole-orange

Peptide bioconjugates combine the molecular recognition features of the polypeptide from a DNA-binding protein and the DNA-sensitive fluorescence of an intercalating dye. Here, DNA template-directed assembly of two bioconjugate probes was examined by steady-state fluorescence resonance energy transfer and time-resolved single photon counting. The Förster critical distance was determined to be approximately 26 A for the oxazole yellow and thiazole orange donor-acceptor pair. The efficiency of energy transfer for two bioconjugates was a function of the number of intervening base pairs between two DNA cognate sites. These probes were sufficiently sensitive to detect sequence dependent curvature and polypeptide induced bending of the DNA. Molecular probes capable of examining spatial aspects of protein complexes at promoter sites could yield important information about the early events in transcription initiation.

Topics: Amino Acid Sequence; Base Sequence; Benzothiazoles; Benzoxazoles; DNA; DNA Nucleotidyltransferases; DNA-Binding Proteins; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Intercalating Agents; Molecular Sequence Data; Peptide Fragments; Peptides; Protein Binding; Quinolines; Spectrometry, Fluorescence; Spectrophotometry; Transposases

The fluorescence induced by polarized light sources, such as the lasers that are used in flow cytometry, is often polarized and anisotropic. In addition, most optical detector systems are sensitive to the direction of polarization. These two factors influence the accuracy of fluorescence intensity measurements. The intensity of two light sources can be compared only if all details of the direction and degree of polarization are known. In a previous study, we observed that fluorescence polarization might be modified by dye-dye interactions. This report further investigates the role of dye density in fluorescence polarization anisotropy.. We measured the polarization distribution of samples stained with commonly used DNA dyes. To determine the role of fluorophore proximity, we compared the monomeric and a dimeric form of the DNA dyes ethidium bromide (EB), thiazole orange (TO), and oxazole yellow (YO).. In all dyes sampled, fluorescence polarization is less at high dye concentrations than at low concentrations. The monomeric dyes exhibit a higher degree of polarization than the dimeric dyes of the same species.. The polarization of fluorescence from DNA dyes is related to the density of incorporation into the DNA helix. Energy transfer between molecules that are in close proximity loosens the linkage between the excitation and emission dipoles, thereby reducing the degree of polarization of the emission.

Topics: Animals; Benzothiazoles; Benzoxazoles; DNA; Fluorescence Polarization; Fluorescent Dyes; Intercalating Agents; Mice; Quinolines; Thiazoles

The monomeric fluorescent dye, SYBR Green I, was investigated and compared with the dyes YO-PRO-1 and thiazole orange (TO) for their application in capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection of double-stranded DNA (dsDNA). DNA fragments were injected by hydrodynamic pressure and separated in a replaceable matrix of hydroxypropyl methylcellulose. For all 3 dyes, optimal concentrations were established and efficient separations of DNA fragments ranging in size from 75 to 12216 bp were obtained. The most promising results in terms of linear detection range were achieved with SYBR Green I. At the optimal dye concentration, fluorescence intensity versus DNA concentration was linear over more than three orders of magnitude (4 pg/microliters to 30 ng/microliters). Limit of detection (LOD) with SYBR Green I was approximately 80 fg of dsDNA (240 zmol of a 200-bp fragment). Similar LOD was obtained with YO-PRO-1, whereas TO resulted in lower sensitivity. Precision in both fluorescence intensity and migration time was high (relative standard deviation, RSD < 3.6%; n = 10) for dsDNA fragments complexed with SYBR Green I. In conclusion, SYBR Green I is fluorescent dye well suited for efficient separation and quantitative, sensitive, and precise determination of dsDNA by CE-LIF.

Topics: Benzothiazoles; Benzoxazoles; Diamines; DNA; Electrophoresis, Capillary; Fluorescent Dyes; Humans; Lasers; Linear Models; Organic Chemicals; Polymerase Chain Reaction; Quinolines; Quinolinium Compounds; Reproducibility of Results; Spectrometry, Fluorescence; Thiazoles