Characterization of hairpin-duplex interconversion of DNA using polyacrylamide gel electrophoresis
Identifieur interne : 000E79 ( Istex/Corpus ); précédent : 000E78; suivant : 000E80Characterization of hairpin-duplex interconversion of DNA using polyacrylamide gel electrophoresis
Auteurs : Michael Shubsda ; Jerry Goodisman ; James C. DabrowiakSource :
- 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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<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title>Characterization of hairpin-duplex interconversion of DNA using polyacrylamide gel electrophoresis</title><author><name sortKey="Shubsda, Michael" sort="Shubsda, Michael" uniqKey="Shubsda M" first="Michael" last="Shubsda">Michael Shubsda</name><affiliation><mods:affiliation>Department of Chemistry, Syracuse University, 1-014 Center for Science and Technology, Syracuse, New York 13244-4100, USA</mods:affiliation></affiliation></author><author><name sortKey="Goodisman, Jerry" sort="Goodisman, Jerry" uniqKey="Goodisman J" first="Jerry" last="Goodisman">Jerry Goodisman</name><affiliation><mods:affiliation>Department of Chemistry, Syracuse University, 1-014 Center for Science and Technology, Syracuse, New York 13244-4100, USA</mods:affiliation></affiliation></author><author><name sortKey="Dabrowiak, James C" sort="Dabrowiak, James C" uniqKey="Dabrowiak J" first="James C." last="Dabrowiak">James C. Dabrowiak</name><affiliation><mods:affiliation>Department of Chemistry, Syracuse University, 1-014 Center for Science and Technology, Syracuse, New York 13244-4100, USA</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:354B0446C04072087A415114AC184A6D78D0066E</idno><date when="1999" year="1999">1999</date><idno type="doi">10.1016/S0301-4622(98)00217-8</idno><idno type="url">https://api.istex.fr/ark:/67375/6H6-XDN99RJS-S/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">000E79</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">000E79</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a">Characterization of hairpin-duplex interconversion of DNA using polyacrylamide gel electrophoresis</title><author><name sortKey="Shubsda, Michael" sort="Shubsda, Michael" uniqKey="Shubsda M" first="Michael" last="Shubsda">Michael Shubsda</name><affiliation><mods:affiliation>Department of Chemistry, Syracuse University, 1-014 Center for Science and Technology, Syracuse, New York 13244-4100, USA</mods:affiliation></affiliation></author><author><name sortKey="Goodisman, Jerry" sort="Goodisman, Jerry" uniqKey="Goodisman J" first="Jerry" last="Goodisman">Jerry Goodisman</name><affiliation><mods:affiliation>Department of Chemistry, Syracuse University, 1-014 Center for Science and Technology, Syracuse, New York 13244-4100, USA</mods:affiliation></affiliation></author><author><name sortKey="Dabrowiak, James C" sort="Dabrowiak, James C" uniqKey="Dabrowiak J" first="James C." last="Dabrowiak">James C. Dabrowiak</name><affiliation><mods:affiliation>Department of Chemistry, Syracuse University, 1-014 Center for Science and Technology, Syracuse, New York 13244-4100, USA</mods:affiliation></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><istex><corpusName>elsevier</corpusName><keywords><teeft><json:string>dimer</json:string><json:string>monomer</json:string><json:string>dimerization</json:string><json:string>ionic strength</json:string><json:string>shubsda</json:string><json:string>biophysical chemistry</json:string><json:string>dimer peaks</json:string><json:string>interconversion</json:string><json:string>radiolabeled</json:string><json:string>rate constants</json:string><json:string>gaussians</json:string><json:string>differential equations</json:string><json:string>ionic strengths</json:string><json:string>oligomer</json:string><json:string>polyacrylamide</json:string><json:string>equilibration</json:string><json:string>intensity patterns</json:string><json:string>electrophoresis time</json:string><json:string>unlabeled</json:string><json:string>broad peak</json:string><json:string>autoradiogram</json:string><json:string>bulk solution</json:string><json:string>ehresmann</json:string><json:string>peak areas</json:string><json:string>kinetics</json:string><json:string>intensity pattern</json:string><json:string>migration velocities</json:string><json:string>biochemistry</json:string><json:string>equilibrating</json:string><json:string>dna</json:string><json:string>total concentration</json:string><json:string>spot intensities</json:string><json:string>relative amounts</json:string><json:string>nucleic acids</json:string><json:string>electrophoresis</json:string><json:string>total intensity</json:string><json:string>monomer concentrations</json:string><json:string>migration velocity</json:string><json:string>peak intensities</json:string><json:string>single peak</json:string><json:string>equilibrating mixture</json:string><json:string>biophysical</json:string><json:string>hairpin</json:string><json:string>peak</json:string><json:string>monomer intensity</json:string><json:string>central peak</json:string><json:string>dimer peak</json:string><json:string>linear background</json:string><json:string>human virus type</json:string><json:string>calculations show</json:string><json:string>chemical reaction</json:string><json:string>electrophoresis distance</json:string><json:string>dimer forms</json:string><json:string>various temperatures</json:string><json:string>single spot</json:string><json:string>other dnas</json:string><json:string>initial dimer</json:string><json:string>dimer spot</json:string><json:string>simulation</json:string><json:string>nucleic</json:string><json:string>second form</json:string><json:string>total volume</json:string><json:string>buffer solution</json:string><json:string>literature values</json:string><json:string>middle peak</json:string><json:string>small fraction</json:string><json:string>homogeneous solution</json:string><json:string>monomer peak</json:string><json:string>dimer spots</json:string><json:string>optical density</json:string><json:string>interconverting mixture</json:string><json:string>equilibrium constants</json:string><json:string>dimerization constants</json:string><json:string>native polyacrylamide</json:string><json:string>electrophoresis experiments</json:string><json:string>fractional intensities</json:string><json:string>electrophoresis times</json:string><json:string>migration</json:string><json:string>ionic</json:string></teeft></keywords><author><json:item><name>Michael Shubsda</name><affiliations><json:string>Department of Chemistry, Syracuse University, 1-014 Center for Science and Technology, Syracuse, New York 13244-4100, USA</json:string></affiliations></json:item><json:item><name>Jerry Goodisman</name><affiliations><json:string>Department of Chemistry, Syracuse University, 1-014 Center for Science and Technology, Syracuse, New York 13244-4100, USA</json:string></affiliations></json:item><json:item><name>James C. Dabrowiak</name><affiliations><json:string>Department of Chemistry, Syracuse University, 1-014 Center for Science and Technology, Syracuse, New York 13244-4100, USA</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>DNA</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Dimerization</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Hairpin</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Duplex</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Electrophoresis</value></json:item></subject><arkIstex>ark:/67375/6H6-XDN99RJS-S</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>Full-length article</json:string></originalGenre><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.</abstract><qualityIndicators><score>10</score><pdfWordCount>10896</pdfWordCount><pdfCharCount>59716</pdfCharCount><pdfVersion>1.2</pdfVersion><pdfPageCount>22</pdfPageCount><pdfPageSize>543 x 743 pts</pdfPageSize><refBibsNative>true</refBibsNative><abstractWordCount>282</abstractWordCount><abstractCharCount>1868</abstractCharCount><keywordCount>5</keywordCount></qualityIndicators><title>Characterization of hairpin-duplex interconversion of DNA using polyacrylamide gel electrophoresis</title><pmid><json:string>11396505</json:string></pmid><pii><json:string>S0301-4622(98)00217-8</json:string></pii><genre><json:string>research-article</json:string></genre><host><title>Biophysical Chemistry</title><language><json:string>unknown</json:string></language><publicationDate>1999</publicationDate><issn><json:string>0301-4622</json:string></issn><pii><json:string>S0301-4622(00)X0047-6</json:string></pii><volume>76</volume><issue>2</issue><pages><first>95</first><last>115</last></pages><genre><json:string>journal</json:string></genre></host><namedEntities><unitex><date><json:string>2000</json:string><json:string>2800</json:string><json:string>1999</json:string><json:string>3000</json:string></date><geogName></geogName><orgName></orgName><orgName_funder></orgName_funder><orgName_provider></orgName_provider><persName><json:string>Ždata</json:string><json:string>In</json:string><json:string>The</json:string><json:string>Michael Shubsda</json:string><json:string>P. N. Borer</json:string><json:string>Jerry GoodismanU</json:string></persName><placeName><json:string>York</json:string></placeName><ref_url></ref_url><ref_bibl><json:string>Kleinschmidt et al.</json:string><json:string>M. M. Shubsda et al.</json:string><json:string>Ross et al.</json:string><json:string>M. Shubsda et al.</json:string><json:string>Yang et al.</json:string></ref_bibl><bibl></bibl></unitex></namedEntities><ark><json:string>ark:/67375/6H6-XDN99RJS-S</json:string></ark><categories><wos><json:string>1 - science</json:string><json:string>2 - chemistry, physical</json:string><json:string>2 - biophysics</json:string><json:string>2 - biochemistry & molecular biology</json:string></wos><scienceMetrix><json:string>1 - health sciences</json:string><json:string>2 - biomedical research</json:string><json:string>3 - biophysics</json:string></scienceMetrix><scopus><json:string>1 - Physical Sciences</json:string><json:string>2 - Chemistry</json:string><json:string>3 - Organic Chemistry</json:string><json:string>1 - Life Sciences</json:string><json:string>2 - Biochemistry, Genetics and Molecular Biology</json:string><json:string>3 - Biochemistry</json:string><json:string>1 - Life Sciences</json:string><json:string>2 - Biochemistry, Genetics and Molecular Biology</json:string><json:string>3 - Biophysics</json:string></scopus><inist><json:string>1 - sciences appliquees, technologies et medecines</json:string><json:string>2 - sciences biologiques et medicales</json:string><json:string>3 - sciences biologiques fondamentales et appliquees. psychologie</json:string></inist></categories><publicationDate>1999</publicationDate><copyrightDate>1999</copyrightDate><doi><json:string>10.1016/S0301-4622(98)00217-8</json:string></doi><id>354B0446C04072087A415114AC184A6D78D0066E</id><score>1</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-XDN99RJS-S/fulltext.pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-XDN99RJS-S/bundle.zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/6H6-XDN99RJS-S/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a">Characterization of hairpin-duplex interconversion of DNA using polyacrylamide gel electrophoresis</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher scheme="https://scientific-publisher.data.istex.fr">ELSEVIER</publisher><availability><licence><p>©1999 Elsevier Science B.V.</p></licence><p scheme="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</p></availability><date>1999</date></publicationStmt><notesStmt><note type="research-article" scheme="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</note><note type="journal" scheme="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</note><note type="content">Fig. 1: Intensity vs. distance at 600 and 3000 sec, from solution of differential Eq. (6)and Eq. (7)with υM=0.015 cm s−1, υD=0.0045 cm s−1, DM=2×10−5 cm2 s−1, DD=6×10−6 cm2s−1, kf=8 M−1 s−1, kr=9.6×10−5 s−1. Initial dimer and monomer concentrations: 6 μM and 3 μM.</note><note type="content">Fig. 2: Intensity vs. distance at 600 and 3000 sec, from solution of differential Eq. (6)and Eq. (7)with υM=0.015 cm s−1, υD=0.0045 cm s−1, DM=2×10−5 cm2 s−1, DD=6×10−6 cm2 s−1, kf=80 M−1 s−1, kr=9.6×10−4 s−1. Initial dimer and monomer concentrations: 6 μM and 3 μM.</note><note type="content">Fig. 3: Intensity vs. distance at 600 and 3000 sec, from solution of differential Eq. (6)and Eq. (7)with υM=0.015 cm s−1, υD=0.0045 cm s−1, DM=2×10−5 cm2 s−1, DD=6×106 cm2 s−1, kf=80 M−1 s−1, kr=9.6×10−4 s−1. Initial dimer and monomer concentrations: 768 μM and 96 μM.</note><note type="content">Fig. 4: Intensity vs. distance at 2000 and 2800 sec, from solution of differential Eq. (6)and Eq. (7)with υM=0.015 cm s−1, υD=0.0045 cm s−1, DM=2×10−5 cm2 s−1, DD=6×10−6 cm2 s−1, kf=20 M−1 s−1, kr=0.0012 s−1. Initial dimer and monomer concentrations: 96 μM and 48 μM.</note><note type="content">Fig. 5: Autoradiogram of electrophoresis gel for 6,7 GA, pre-equilibrated at temperatures of 90°C (lanes 1–2), 56°C (lanes 3–4), 37°C (lanes 5–6), 25°C (lanes 7–8), and 5°C (lanes 9–10). The odd numbered lanes have ionic strength of 190 mM and even numbered lanes have I=97 mM. Since the direction of migration is from top to bottom, the lower spot in each lane is monomer, and the higher is dimer.</note><note type="content">Fig. 6: Optical density as a function of electrophoresis distance for 6,7 GA equilibrated at various temperatures before loading into 5°C gel. Ionic strength=190 mM. From top to bottom, equilibration temperatures are: 90°C, 56°C, 37°C, 25°C, 5°C.</note><note type="content">Fig. 7: Autoradiogram of load-and-run gel for WC DNA at 0.287 μM, ionic strength 190 mM in odd numbered lanes and 100 mM in even numbered lanes. In each lane, lower spot is monomer, upper spot is dimer. Electrophoresis times in h: 5 (lanes 1–2), 4.5 (3–4), 3.5 (5–6), 2.5 (7–8), 1.5 (9–10), 0.75 (11–12), 0.25 (13–14).</note><note type="content">Fig. 8: Optical density as a function of electrophoresis distance for load-and-run gel for WC DNA; (a) running times, in h, are (top to bottom): 0.25, 0.75, 1.5, 2.5. (b) running times, in h, are (top to bottom): 2.5, 3.5, 4.5, 5.</note><note type="content">Fig. 9: Autoradiogram of WC DNA gel. The concentrations of the unlabeled DNA, before correction for dilution in the well, are 1.875 μM (lanes 1–2), 0.75 μM (lanes 3–4), 0.375 μM (lanes 5–6), 0.075 μM (lanes 7–8), 0.052 μM (lane 9–10), 0.030 μM (lanes 11–12), and 0 μM (lanes 13–14). Electrophoresis time=5 h, gel temperature=5°C. Alternate lanes are for ionic strengths 97 and 190 mM (right to left).</note><note type="content">Fig. 10: Measured values of KC plotted vs. DNA concentration in μM and linear fits. The slope gives the true value of K and the x-intercept the negative of the concentration of DNA added with the radiolabeled DNA.</note><note type="content">Table 1: Calculation of apparent K for determination of labeled DNA concentration</note><note type="content">Table 2: Peak areasa for 6,7 GA, and KC values calculated from two models</note><note type="content">Table 3: Peak areasa for 6,7 GA run at various concentrations and calculated values for KC and K</note><note type="content">Table 4: Load and run results for 6,7GA at 5°C</note><note type="content">Table 5: Load and run results for 6,7 GA at 0.49 μM, 5°C</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a">Characterization of hairpin-duplex interconversion of DNA using polyacrylamide gel electrophoresis</title><author xml:id="author-0000"><persName><forename type="first">Michael</forename><surname>Shubsda</surname></persName><affiliation>Department of Chemistry, Syracuse University, 1-014 Center for Science and Technology, Syracuse, New York 13244-4100, USA</affiliation></author><author xml:id="author-0001"><persName><forename type="first">Jerry</forename><surname>Goodisman</surname></persName><note type="correspondence"><p>Corresponding author.</p></note><affiliation>Department of Chemistry, Syracuse University, 1-014 Center for Science and Technology, Syracuse, New York 13244-4100, USA</affiliation></author><author xml:id="author-0002"><persName><forename type="first">James C.</forename><surname>Dabrowiak</surname></persName><affiliation>Department of Chemistry, Syracuse University, 1-014 Center for Science and Technology, Syracuse, New York 13244-4100, USA</affiliation></author><idno type="istex">354B0446C04072087A415114AC184A6D78D0066E</idno><idno type="ark">ark:/67375/6H6-XDN99RJS-S</idno><idno type="DOI">10.1016/S0301-4622(98)00217-8</idno><idno type="PII">S0301-4622(98)00217-8</idno></analytic><monogr><title level="j">Biophysical Chemistry</title><title level="j" type="abbrev">BIOCHE</title><idno type="pISSN">0301-4622</idno><idno type="PII">S0301-4622(00)X0047-6</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="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></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>1999</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>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.</p></abstract><textClass><keywords scheme="keyword"><list><head>Keywords</head><item><term>DNA</term></item><item><term>Dimerization</term></item><item><term>Hairpin</term></item><item><term>Duplex</term></item><item><term>Electrophoresis</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="1998-10-30">Modified</change><change when="1999">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-XDN99RJS-S/fulltext.txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Elsevier, elements deleted: ce:floats; body; tail"><istex:xmlDeclaration>version="1.0" encoding="utf-8"</istex:xmlDeclaration><istex:docType PUBLIC="-//ES//DTD journal article DTD version 4.5.2//EN//XML" URI="art452.dtd" name="istex:docType"><istex:entity SYSTEM="gr1" NDATA="IMAGE" name="gr1"></istex:entity><istex:entity SYSTEM="gr2" NDATA="IMAGE" name="gr2"></istex:entity><istex:entity SYSTEM="gr3" NDATA="IMAGE" name="gr3"></istex:entity><istex:entity SYSTEM="gr4" NDATA="IMAGE" name="gr4"></istex:entity><istex:entity SYSTEM="gr5" NDATA="IMAGE" name="gr5"></istex:entity><istex:entity SYSTEM="gr6" NDATA="IMAGE" name="gr6"></istex:entity><istex:entity SYSTEM="gr7" NDATA="IMAGE" name="gr7"></istex:entity><istex:entity SYSTEM="gr8" NDATA="IMAGE" name="gr8"></istex:entity><istex:entity SYSTEM="gr9" NDATA="IMAGE" name="gr9"></istex:entity><istex:entity SYSTEM="gr10" NDATA="IMAGE" name="gr10"></istex:entity></istex:docType><istex:document><converted-article version="4.5.2" docsubtype="fla"><item-info><jid>BIOCHE</jid><aid>2525</aid><ce:pii>S0301-4622(98)00217-8</ce:pii><ce:doi>10.1016/S0301-4622(98)00217-8</ce:doi><ce:copyright year="1999" type="full-transfer">Elsevier Science B.V.</ce:copyright></item-info><head><ce:title>Characterization of hairpin-duplex interconversion of DNA using polyacrylamide gel electrophoresis</ce:title><ce:author-group><ce:author><ce:given-name>Michael</ce:given-name><ce:surname>Shubsda</ce:surname></ce:author><ce:author><ce:given-name>Jerry</ce:given-name><ce:surname>Goodisman</ce:surname><ce:cross-ref refid="CORR1">*</ce:cross-ref></ce:author><ce:author><ce:given-name>James C.</ce:given-name><ce:surname>Dabrowiak</ce:surname></ce:author><ce:affiliation><ce:textfn>Department of Chemistry, Syracuse University, 1-014 Center for Science and Technology, Syracuse, New York 13244-4100, USA</ce:textfn></ce:affiliation><ce:correspondence id="CORR1"><ce:label>*</ce:label><ce:text>Corresponding author.</ce:text></ce:correspondence></ce:author-group><ce:date-received day="29" month="9" year="1998"></ce:date-received><ce:date-revised day="30" month="10" year="1998"></ce:date-revised><ce:date-accepted day="30" month="10" year="1998"></ce:date-accepted><ce:abstract><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para>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.</ce:simple-para></ce:abstract-sec></ce:abstract><ce:keywords class="keyword"><ce:section-title>Keywords</ce:section-title><ce:keyword><ce:text>DNA</ce:text></ce:keyword><ce:keyword><ce:text>Dimerization</ce:text></ce:keyword><ce:keyword><ce:text>Hairpin</ce:text></ce:keyword><ce:keyword><ce:text>Duplex</ce:text></ce:keyword><ce:keyword><ce:text>Electrophoresis</ce:text></ce:keyword></ce:keywords></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo><title>Characterization of hairpin-duplex interconversion of DNA using polyacrylamide gel electrophoresis</title></titleInfo><titleInfo type="alternative" contentType="CDATA"><title>Characterization of hairpin-duplex interconversion of DNA using polyacrylamide gel electrophoresis</title></titleInfo><name type="personal"><namePart type="given">Michael</namePart><namePart type="family">Shubsda</namePart><affiliation>Department of Chemistry, Syracuse University, 1-014 Center for Science and Technology, Syracuse, New York 13244-4100, USA</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Jerry</namePart><namePart type="family">Goodisman</namePart><affiliation>Department of Chemistry, Syracuse University, 1-014 Center for Science and Technology, Syracuse, New York 13244-4100, USA</affiliation><description>Corresponding author.</description><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">James C.</namePart><namePart type="family">Dabrowiak</namePart><affiliation>Department of Chemistry, Syracuse University, 1-014 Center for Science and Technology, Syracuse, New York 13244-4100, USA</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="Full-length article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">1999</dateIssued><dateModified encoding="w3cdtf">1998-10-30</dateModified><copyrightDate encoding="w3cdtf">1999</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract 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.</abstract><note type="content">Fig. 1: Intensity vs. distance at 600 and 3000 sec, from solution of differential Eq. (6)and Eq. (7)with υM=0.015 cm s−1, υD=0.0045 cm s−1, DM=2×10−5 cm2 s−1, DD=6×10−6 cm2s−1, kf=8 M−1 s−1, kr=9.6×10−5 s−1. Initial dimer and monomer concentrations: 6 μM and 3 μM.</note><note type="content">Fig. 2: Intensity vs. distance at 600 and 3000 sec, from solution of differential Eq. (6)and Eq. (7)with υM=0.015 cm s−1, υD=0.0045 cm s−1, DM=2×10−5 cm2 s−1, DD=6×10−6 cm2 s−1, kf=80 M−1 s−1, kr=9.6×10−4 s−1. Initial dimer and monomer concentrations: 6 μM and 3 μM.</note><note type="content">Fig. 3: Intensity vs. distance at 600 and 3000 sec, from solution of differential Eq. (6)and Eq. (7)with υM=0.015 cm s−1, υD=0.0045 cm s−1, DM=2×10−5 cm2 s−1, DD=6×106 cm2 s−1, kf=80 M−1 s−1, kr=9.6×10−4 s−1. Initial dimer and monomer concentrations: 768 μM and 96 μM.</note><note type="content">Fig. 4: Intensity vs. distance at 2000 and 2800 sec, from solution of differential Eq. (6)and Eq. (7)with υM=0.015 cm s−1, υD=0.0045 cm s−1, DM=2×10−5 cm2 s−1, DD=6×10−6 cm2 s−1, kf=20 M−1 s−1, kr=0.0012 s−1. Initial dimer and monomer concentrations: 96 μM and 48 μM.</note><note type="content">Fig. 5: Autoradiogram of electrophoresis gel for 6,7 GA, pre-equilibrated at temperatures of 90°C (lanes 1–2), 56°C (lanes 3–4), 37°C (lanes 5–6), 25°C (lanes 7–8), and 5°C (lanes 9–10). The odd numbered lanes have ionic strength of 190 mM and even numbered lanes have I=97 mM. Since the direction of migration is from top to bottom, the lower spot in each lane is monomer, and the higher is dimer.</note><note type="content">Fig. 6: Optical density as a function of electrophoresis distance for 6,7 GA equilibrated at various temperatures before loading into 5°C gel. Ionic strength=190 mM. From top to bottom, equilibration temperatures are: 90°C, 56°C, 37°C, 25°C, 5°C.</note><note type="content">Fig. 7: Autoradiogram of load-and-run gel for WC DNA at 0.287 μM, ionic strength 190 mM in odd numbered lanes and 100 mM in even numbered lanes. In each lane, lower spot is monomer, upper spot is dimer. Electrophoresis times in h: 5 (lanes 1–2), 4.5 (3–4), 3.5 (5–6), 2.5 (7–8), 1.5 (9–10), 0.75 (11–12), 0.25 (13–14).</note><note type="content">Fig. 8: Optical density as a function of electrophoresis distance for load-and-run gel for WC DNA; (a) running times, in h, are (top to bottom): 0.25, 0.75, 1.5, 2.5. (b) running times, in h, are (top to bottom): 2.5, 3.5, 4.5, 5.</note><note type="content">Fig. 9: Autoradiogram of WC DNA gel. The concentrations of the unlabeled DNA, before correction for dilution in the well, are 1.875 μM (lanes 1–2), 0.75 μM (lanes 3–4), 0.375 μM (lanes 5–6), 0.075 μM (lanes 7–8), 0.052 μM (lane 9–10), 0.030 μM (lanes 11–12), and 0 μM (lanes 13–14). Electrophoresis time=5 h, gel temperature=5°C. Alternate lanes are for ionic strengths 97 and 190 mM (right to left).</note><note type="content">Fig. 10: Measured values of KC plotted vs. DNA concentration in μM and linear fits. The slope gives the true value of K and the x-intercept the negative of the concentration of DNA added with the radiolabeled DNA.</note><note type="content">Table 1: Calculation of apparent K for determination of labeled DNA concentration</note><note type="content">Table 2: Peak areasa for 6,7 GA, and KC values calculated from two models</note><note type="content">Table 3: Peak areasa for 6,7 GA run at various concentrations and calculated values for KC and K</note><note type="content">Table 4: Load and run results for 6,7GA at 5°C</note><note type="content">Table 5: Load and run results for 6,7 GA at 0.49 μM, 5°C</note><subject><genre>Keywords</genre><topic>DNA</topic><topic>Dimerization</topic><topic>Hairpin</topic><topic>Duplex</topic><topic>Electrophoresis</topic></subject><relatedItem type="host"><titleInfo><title>Biophysical Chemistry</title></titleInfo><titleInfo type="abbreviated"><title>BIOCHE</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">1999</dateIssued></originInfo><identifier type="ISSN">0301-4622</identifier><identifier type="PII">S0301-4622(00)X0047-6</identifier><part><date>1999</date><detail type="volume"><number>76</number><caption>vol.</caption></detail><detail type="issue"><number>2</number><caption>no.</caption></detail><extent unit="issue-pages"><start>87</start><end>160</end></extent><extent unit="pages"><start>95</start><end>115</end></extent></part></relatedItem><identifier type="istex">354B0446C04072087A415114AC184A6D78D0066E</identifier><identifier type="ark">ark:/67375/6H6-XDN99RJS-S</identifier><identifier type="DOI">10.1016/S0301-4622(98)00217-8</identifier><identifier type="PII">S0301-4622(98)00217-8</identifier><accessCondition type="use and reproduction" contentType="copyright">©1999 Elsevier Science B.V.</accessCondition><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</recordContentSource><recordOrigin>Elsevier Science B.V., ©1999</recordOrigin></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-XDN99RJS-S/record.json</uri></json:item></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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