Characterization of hairpin-duplex interconversion of DNA using polyacrylamide gel electrophoresis
Identifieur interne : 000E79 ( Istex/Curation ); précédent : 000E78; suivant : 000E80Characterization of hairpin-duplex interconversion of DNA using polyacrylamide gel electrophoresis
Auteurs : Michael Shubsda [États-Unis] ; Jerry Goodisman [États-Unis] ; James C. Dabrowiak [États-Unis]Source :
- Biophysical Chemistry [ 0301-4622 ] ; 1999.
English descriptors
- Teeft :
- Autoradiogram, Biochemistry, Biophysical, Biophysical chemistry, Broad peak, Bulk solution, Calculations show, Central peak, Chemical reaction, Differential equations, Dimer, Dimer forms, Dimer peak, Dimer peaks, Dimer spot, Dimerization, Dna, Ehresmann, Electrophoresis, Electrophoresis distance, Electrophoresis time, Equilibrating, Equilibrating mixture, Equilibration, Gaussians, Hairpin, Human virus type, Initial dimer, Intensity pattern, Intensity patterns, Interconversion, Ionic strength, Ionic strengths, Kinetics, Linear background, Migration velocities, Migration velocity, Monomer, Monomer concentrations, Monomer intensity, Nucleic, Nucleic acids, Oligomer, Other dnas, Peak, Peak areas, Peak intensities, Polyacrylamide, Radiolabeled, Rate constants, Relative amounts, Shubsda, Simulation, Single peak, Single spot, Spot intensities, Total concentration, Total intensity, Unlabeled, Various temperatures.
Abstract
Abstract: We show how polyacrylamide gel electrophoresis of radiolabeled DNA can be used to measure the hairpin-duplex equilibrium constant for DNA in solution. As an aid to the interpretation of the experiments, the differential equations associated with diffusion, migration and chemical reaction of the DNA forms are solved and intensity patterns generated. Two kinds of experiments were performed on several DNA 12-mers: in the first, electrophoresis time was constant while DNA concentration varied; in the other, concentration was constant while time varied (a `load-and-run' gel). The observed patterns depended on the gel temperature and not the temperature at which the DNA was equilibrated before loading in the well, because reequilibration occurs before the DNA leaves the well to enter the gel proper. During this time, mixing also occurs, changing the concentration and ionic strength of the sample. A method of calculating the true DNA concentration, including the unmeasured concentration added with the radiolabel, is given. When the intensity pattern consists mainly of monomer and dimer peaks, the equilibrium constant K is easily calculated from peak intensities. However, when there is significant intensity between the peaks (which the calculations show results from monomer–dimer interconversion in the gel), K will be inaccurate. An accurate value of K may be determined from a load-and-run gel by extrapolating back to time 0. When the intensity pattern consists of a single broad peak (from rapid monomer-dimer interconversion in the gel), K cannot be calculated without additional information. The rate of interconversion increases with temperature. Estimated rates in the gel are more than an order of magnitude smaller than in bulk solution at the same temperature. Derived values of K for several DNAs are compared with literature values.
Url:
DOI: 10.1016/S0301-4622(98)00217-8
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Dabrowiak</name><affiliation wicri:level="1"><mods:affiliation>Department of Chemistry, Syracuse University, 1-014 Center for Science and Technology, Syracuse, New York 13244-4100, USA</mods:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Chemistry, Syracuse University, 1-014 Center for Science and Technology, Syracuse, New York 13244-4100</wicri:regionArea></affiliation></author></analytic><monogr></monogr><series><title level="j">Biophysical Chemistry</title><title level="j" type="abbrev">BIOCHE</title><idno type="ISSN">0301-4622</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1999">1999</date><biblScope unit="volume">76</biblScope><biblScope unit="issue">2</biblScope><biblScope unit="page" from="95">95</biblScope><biblScope unit="page" to="115">115</biblScope></imprint><idno type="ISSN">0301-4622</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0301-4622</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="Teeft" xml:lang="en"><term>Autoradiogram</term><term>Biochemistry</term><term>Biophysical</term><term>Biophysical chemistry</term><term>Broad peak</term><term>Bulk solution</term><term>Calculations show</term><term>Central peak</term><term>Chemical reaction</term><term>Differential equations</term><term>Dimer</term><term>Dimer forms</term><term>Dimer peak</term><term>Dimer peaks</term><term>Dimer spot</term><term>Dimerization</term><term>Dna</term><term>Ehresmann</term><term>Electrophoresis</term><term>Electrophoresis distance</term><term>Electrophoresis time</term><term>Equilibrating</term><term>Equilibrating mixture</term><term>Equilibration</term><term>Gaussians</term><term>Hairpin</term><term>Human virus type</term><term>Initial dimer</term><term>Intensity pattern</term><term>Intensity patterns</term><term>Interconversion</term><term>Ionic strength</term><term>Ionic strengths</term><term>Kinetics</term><term>Linear background</term><term>Migration velocities</term><term>Migration velocity</term><term>Monomer</term><term>Monomer concentrations</term><term>Monomer intensity</term><term>Nucleic</term><term>Nucleic acids</term><term>Oligomer</term><term>Other dnas</term><term>Peak</term><term>Peak areas</term><term>Peak intensities</term><term>Polyacrylamide</term><term>Radiolabeled</term><term>Rate constants</term><term>Relative amounts</term><term>Shubsda</term><term>Simulation</term><term>Single peak</term><term>Single spot</term><term>Spot intensities</term><term>Total concentration</term><term>Total intensity</term><term>Unlabeled</term><term>Various temperatures</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract: We show how polyacrylamide gel electrophoresis of radiolabeled DNA can be used to measure the hairpin-duplex equilibrium constant for DNA in solution. As an aid to the interpretation of the experiments, the differential equations associated with diffusion, migration and chemical reaction of the DNA forms are solved and intensity patterns generated. Two kinds of experiments were performed on several DNA 12-mers: in the first, electrophoresis time was constant while DNA concentration varied; in the other, concentration was constant while time varied (a `load-and-run' gel). The observed patterns depended on the gel temperature and not the temperature at which the DNA was equilibrated before loading in the well, because reequilibration occurs before the DNA leaves the well to enter the gel proper. During this time, mixing also occurs, changing the concentration and ionic strength of the sample. A method of calculating the true DNA concentration, including the unmeasured concentration added with the radiolabel, is given. When the intensity pattern consists mainly of monomer and dimer peaks, the equilibrium constant K is easily calculated from peak intensities. However, when there is significant intensity between the peaks (which the calculations show results from monomer–dimer interconversion in the gel), K will be inaccurate. An accurate value of K may be determined from a load-and-run gel by extrapolating back to time 0. When the intensity pattern consists of a single broad peak (from rapid monomer-dimer interconversion in the gel), K cannot be calculated without additional information. The rate of interconversion increases with temperature. Estimated rates in the gel are more than an order of magnitude smaller than in bulk solution at the same temperature. Derived values of K for several DNAs are compared with literature values.</div></front></TEI></record>Pour manipuler ce document sous Unix (Dilib)
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