tetramethylrhodamine and deoxyuridine-triphosphate

We present results of an approach in which low-density labeled DNA itself provides an amplification of the cross-correlated fluorescent signal in the two-color cross-correlation function. Tetramethylrhodamine-4-dUTP and Cy5-dCTP are incorporated by polymerase chain reaction at multiple positions of the same 217 bp target DNA. We call this novel approach the 'two-color FCS signal amplification'. The signal amplification is an example for interactions of two ligands with different colors at multiple positions of the same target.

Topics: Carbocyanines; Deoxyuracil Nucleotides; DNA; Fluorescent Dyes; Models, Theoretical; Polymerase Chain Reaction; Rhodamines; Spectrometry, Fluorescence

A solution-phase steady-state polarization-based method for discriminating among the four DNA nucleotides labeled identically with tetramethylrhodamine is described and demonstrated. Labeled nucleotides were dissolved in buffered surfactant solutions. In room temperature 4.5 mM Triton X-100 solutions at neutral pH, the measured steady-state polarizations of tetramethylrhodamine-labeled dATP, dCTP, dGTP and dUTP were 0.261 +/- 0.003, 0.112 +/- 0.003, 0.288 +/- 0.003, and 0.147 +/- 0.003, respectively. A blind test of 40 samples showed no errors in classification based on polarization. The reproducibility obtained during this study demonstrates that the four dye-labeled nucleotides can be discriminated with more than 99.8% confidence.

Topics: Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyuracil Nucleotides; Fluorescence Polarization; Fluorescent Dyes; Molecular Structure; Nucleotides; Octoxynol; Rhodamines; Solutions; Surface-Active Agents

We show that fluorescence correlation spectroscopy (FCS) can be used as a reliable, simple, and fast tool for detecting products of the polymerase chain reaction (PCR). By use of autocorrelation experiments, it is demonstrated that fluorescent 217-bp DNA fragments can be detected at very low initial ss M13mp18(+) DNA and tetramethylrhodamine-4-dUTP concentrations and that these polymers are cleaved by the chosen restriction enzymes. A FCS calibration curve is presented, where the translational diffusion times of different size DNA fragments are plotted versus the number of base pairs they contain. At zero and very low template concentrations a large "background" species emerges, which is a reflection of the experimental conditions chosen and the extremely high sensitivity of FCS. The relative amount of nonspecific product formation is less than 1%. The ease by which a FCS measurement can be performed (a few minutes at most) also enables the technique to be used as an effective screening method.

Topics: Base Composition; Calibration; Deoxyribonucleases; Deoxyuracil Nucleotides; DNA, Single-Stranded; Fluorescence Polarization; Fluorescent Dyes; Polymerase Chain Reaction; Polymers; Quantum Theory; Rhodamines; Spectrometry, Fluorescence

Optimal sensitivity of fluorescence in situ hybridization (FISH) requires bright signals and low background fluorescence. Use of locus-specific probes is especially dependent on high sensitivity. Some tissue preparations show high autofluorescence, masking small or dim signals. We have developed a new method for subtracting autofluorescence from digital images on a pixel-by-pixel basis. It is based on the observation that fluorescent labels for FISH have narrower excitation and emission spectra than the chemical components responsible for autofluorescence. Our new approach uses calculation of the ratio of autofluorescence between multiple color images for correction of autofluorescence in each individual image. By subtracting autofluorescence components, we were able to enhance centromeric signals and make previously indistinguishable cosmid signals clearly visible. This image-processing approach to autofluorescence correction may widen the applicability of gene-specific probes in FISH analysis of tumor material.

Topics: Artifacts; Centromere; Chromosomes, Human, Pair 17; Deoxyuracil Nucleotides; Digoxigenin; DNA Probes; DNA, Neoplasm; Fluorescein; Fluoresceins; Fluorescence; Fluorescent Dyes; Humans; In Situ Hybridization, Fluorescence; Neoplasm Proteins; Receptor, ErbB-2; Rhodamines; Subtraction Technique; Urinary Bladder Neoplasms