Fluorescence correlation spectroscopy of enzymatic DNA polymerization. 1998

S Björling, and M Kinjo, and Z Földes-Papp, and E Hagman, and P Thyberg, and R Rigler
Department of Medical Biophysics, MBB, Karolinska Institute, Stockholm, Sweden.

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.

UI MeSH Term Description Entries
D011108 Polymers Compounds formed by the joining of smaller, usually repeating, units linked by covalent bonds. These compounds often form large macromolecules (e.g., BIOPOLYMERS; PLASTICS). Polymer
D011789 Quantum Theory The theory that the radiation and absorption of energy take place in definite quantities called quanta (E) which vary in size and are defined by the equation E Quantum Theories,Theories, Quantum,Theory, Quantum
D002138 Calibration Determination, by measurement or comparison with a standard, of the correct value of each scale reading on a meter or other measuring instrument; or determination of the settings of a control device that correspond to particular values of voltage, current, frequency or other output. Calibrations
D003851 Deoxyribonucleases Enzymes which catalyze the hydrolases of ester bonds within DNA. EC 3.1.-. DNAase,DNase,Deoxyribonuclease,Desoxyribonuclease,Desoxyribonucleases,Nucleases, DNA,Acid DNase,Alkaline DNase,DNA Nucleases,DNase, Acid,DNase, Alkaline
D003856 Deoxyuracil Nucleotides Uracil nucleotides which contain deoxyribose as the sugar moiety. Deoxyuridine Phosphates,Nucleotides, Deoxyuracil,Phosphates, Deoxyuridine
D004277 DNA, Single-Stranded A single chain of deoxyribonucleotides that occurs in some bacteria and viruses. It usually exists as a covalently closed circle. Single-Stranded DNA,DNA, Single Stranded,Single Stranded DNA
D005454 Fluorescence Polarization Measurement of the polarization of fluorescent light from solutions or microscopic specimens. It is used to provide information concerning molecular size, shape, and conformation, molecular anisotropy, electronic energy transfer, molecular interaction, including dye and coenzyme binding, and the antigen-antibody reaction. Anisotropy, Fluorescence,Fluorescence Anisotropy,Polarization, Fluorescence,Anisotropies, Fluorescence,Fluorescence Anisotropies,Fluorescence Polarizations,Polarizations, Fluorescence
D005456 Fluorescent Dyes Chemicals that emit light after excitation by light. The wave length of the emitted light is usually longer than that of the incident light. Fluorochromes are substances that cause fluorescence in other substances, i.e., dyes used to mark or label other compounds with fluorescent tags. Flourescent Agent,Fluorescent Dye,Fluorescent Probe,Fluorescent Probes,Fluorochrome,Fluorochromes,Fluorogenic Substrates,Fluorescence Agents,Fluorescent Agents,Fluorogenic Substrate,Agents, Fluorescence,Agents, Fluorescent,Dyes, Fluorescent,Probes, Fluorescent,Substrates, Fluorogenic
D001482 Base Composition The relative amounts of the PURINES and PYRIMIDINES in a nucleic acid. Base Ratio,G+C Composition,Guanine + Cytosine Composition,G+C Content,GC Composition,GC Content,Guanine + Cytosine Content,Base Compositions,Base Ratios,Composition, Base,Composition, G+C,Composition, GC,Compositions, Base,Compositions, G+C,Compositions, GC,Content, G+C,Content, GC,Contents, G+C,Contents, GC,G+C Compositions,G+C Contents,GC Compositions,GC Contents,Ratio, Base,Ratios, Base
D012235 Rhodamines A family of 3,6-di(substituted-amino)-9-benzoate derivatives of xanthene that are used as dyes and as indicators for various metals; also used as fluorescent tracers in histochemistry. Rhodamine

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