Ligation of Eco RI endonuclease-generated DNA fragments into linear and circular structures
Identifieur interne : 000165 ( Istex/Corpus ); précédent : 000164; suivant : 000166Ligation of Eco RI endonuclease-generated DNA fragments into linear and circular structures
Auteurs : Achilles Dugaiczyk ; Herbert W. Boyer ; Howard M. GoodmanSource :
- Journal of Molecular Biology [ 0022-2836 ] ; 1975.
English descriptors
- Teeft :
- Agarose, Circular forms, Circular molecules, Circular products, Circular structures, Cohesive ends, Contour length, Covalently, Dimer, Dugaiczyk, Ecori, Endonuclease, Fragment, Helling, Ionic strength, Ligase, Ligated, Ligation, Ligation products, Linear concatemers, Linear dimers, Linear form, Linear monomer, Linear monomers, Molecular weight, Molecular weights, Monomer, Monomeric, Open circle, Optimum temperature, Phage, Polynucleotide, Polynucleotide ligase, Present work, Proc, Psclo1, Ring closure, Same molecule, Satellite bands, Superhelical, Superhelical form, Total concentration.
Abstract
Abstract: Double-stranded DNA fragments terminated at their 5′-ends by the singlestranded sequence pA-A-T-T-, generated by digestion of DNA with EcoRI restriction endonuclease, were ligated with Escherichia coli polynucleotide ligase under various conditions of temperature, concentration and time. The linear and circular products of ligation were separated by electrophoresis in agarose gel and quantitated by densitometry. The rate of ligation of (EcoRI-cleaved) simian virus (SV40) DNA at a concentration of 100 μg/ml increased from 0 °C to 5 °C to 10 °C (6-fold increase overall); raising the temperature to 15 °C did not further increase the rate of ligation. At the appropriate DNA concentrations, the predominant products of ligation are either linear concatemers that are integral multimers of the starting DNA fragment, or covalently closed circular structures of the monomeric DNA fragment. Ligating a mixture of two different length DNA fragments gives rise to all of the possible expected recombinant molecules. Linear or circular products of ligation were predicted by consideration of the total concentration of DNA termini, i, and the local concentration of one terminus in the neighborhood of the other on the same DNA molecule, j. The parameter j is a function of the length of a DNA molecule, providing this length is greater than the random coil segment of DNA. Experimentally it was found that circular structures are formed in significant amounts only under conditions when the value of j is several times greater than that of i. When j = i, equal amounts of linear and circular products would be expected, but most of the molecules were ligated into linear concatemers. No circular structure of a DNA fragment whose contour length l (6 × 10−2 μm) is smaller than the random coil segment value b (7·17 × 10−2 μm) was observed, while circular structures of the dimer of the same molecule (12 × 10−2 μm) were detected.
Url:
DOI: 10.1016/0022-2836(75)90189-8
Links to Exploration step
ISTEX:95F72D731B104A5926D19504CA826E84B3E41529Le document en format XML
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Goodman</name><affiliation><mods:affiliation>Department of Biochemistry and Biophysics University of California School of Medicine, San Francisco Calif. 94143, U.S.A.</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">Journal of Molecular Biology</title><title level="j" type="abbrev">YJMBI</title><idno type="ISSN">0022-2836</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1975">1975</date><biblScope unit="volume">96</biblScope><biblScope unit="issue">1</biblScope><biblScope unit="page" from="171">171</biblScope><biblScope unit="page" to="178">178</biblScope></imprint><idno type="ISSN">0022-2836</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0022-2836</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="Teeft" xml:lang="en"><term>Agarose</term><term>Circular forms</term><term>Circular molecules</term><term>Circular products</term><term>Circular structures</term><term>Cohesive ends</term><term>Contour length</term><term>Covalently</term><term>Dimer</term><term>Dugaiczyk</term><term>Ecori</term><term>Endonuclease</term><term>Fragment</term><term>Helling</term><term>Ionic strength</term><term>Ligase</term><term>Ligated</term><term>Ligation</term><term>Ligation products</term><term>Linear concatemers</term><term>Linear dimers</term><term>Linear form</term><term>Linear monomer</term><term>Linear monomers</term><term>Molecular weight</term><term>Molecular weights</term><term>Monomer</term><term>Monomeric</term><term>Open circle</term><term>Optimum temperature</term><term>Phage</term><term>Polynucleotide</term><term>Polynucleotide ligase</term><term>Present work</term><term>Proc</term><term>Psclo1</term><term>Ring closure</term><term>Same molecule</term><term>Satellite bands</term><term>Superhelical</term><term>Superhelical form</term><term>Total concentration</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract: Double-stranded DNA fragments terminated at their 5′-ends by the singlestranded sequence pA-A-T-T-, generated by digestion of DNA with EcoRI restriction endonuclease, were ligated with Escherichia coli polynucleotide ligase under various conditions of temperature, concentration and time. The linear and circular products of ligation were separated by electrophoresis in agarose gel and quantitated by densitometry. The rate of ligation of (EcoRI-cleaved) simian virus (SV40) DNA at a concentration of 100 μg/ml increased from 0 °C to 5 °C to 10 °C (6-fold increase overall); raising the temperature to 15 °C did not further increase the rate of ligation. At the appropriate DNA concentrations, the predominant products of ligation are either linear concatemers that are integral multimers of the starting DNA fragment, or covalently closed circular structures of the monomeric DNA fragment. Ligating a mixture of two different length DNA fragments gives rise to all of the possible expected recombinant molecules. Linear or circular products of ligation were predicted by consideration of the total concentration of DNA termini, i, and the local concentration of one terminus in the neighborhood of the other on the same DNA molecule, j. The parameter j is a function of the length of a DNA molecule, providing this length is greater than the random coil segment of DNA. Experimentally it was found that circular structures are formed in significant amounts only under conditions when the value of j is several times greater than that of i. When j = i, equal amounts of linear and circular products would be expected, but most of the molecules were ligated into linear concatemers. No circular structure of a DNA fragment whose contour length l (6 × 10−2 μm) is smaller than the random coil segment value b (7·17 × 10−2 μm) was observed, while circular structures of the dimer of the same molecule (12 × 10−2 μm) were detected.</div></front></TEI><istex><corpusName>elsevier</corpusName><keywords><teeft><json:string>ligation</json:string><json:string>ecori</json:string><json:string>ligase</json:string><json:string>psclo1</json:string><json:string>dimer</json:string><json:string>molecular weight</json:string><json:string>ligated</json:string><json:string>agarose</json:string><json:string>circular forms</json:string><json:string>dugaiczyk</json:string><json:string>proc</json:string><json:string>monomeric</json:string><json:string>covalently</json:string><json:string>endonuclease</json:string><json:string>polynucleotide</json:string><json:string>phage</json:string><json:string>fragment</json:string><json:string>helling</json:string><json:string>contour length</json:string><json:string>circular structures</json:string><json:string>superhelical</json:string><json:string>circular products</json:string><json:string>molecular weights</json:string><json:string>linear monomer</json:string><json:string>total concentration</json:string><json:string>cohesive ends</json:string><json:string>circular molecules</json:string><json:string>ionic strength</json:string><json:string>monomer</json:string><json:string>satellite bands</json:string><json:string>linear concatemers</json:string><json:string>linear form</json:string><json:string>present work</json:string><json:string>polynucleotide ligase</json:string><json:string>open circle</json:string><json:string>superhelical form</json:string><json:string>same molecule</json:string><json:string>linear dimers</json:string><json:string>ring closure</json:string><json:string>optimum temperature</json:string><json:string>ligation products</json:string><json:string>linear monomers</json:string></teeft></keywords><author><json:item><name>Achilles Dugaiczyk</name><affiliations><json:string>Department of Biochemistry and Biophysics University of California School of Medicine, San Francisco Calif. 94143, U.S.A.</json:string></affiliations></json:item><json:item><name>Herbert W. Boyer</name><affiliations><json:string>Department of Microbiology University of California School of Medicine, San Francisco Calif. 94143, U.S.A.</json:string></affiliations></json:item><json:item><name>Howard M. Goodman</name><affiliations><json:string>Department of Biochemistry and Biophysics University of California School of Medicine, San Francisco Calif. 94143, U.S.A.</json:string></affiliations></json:item></author><arkIstex>ark:/67375/6H6-N6XS16BC-0</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>Full-length article</json:string></originalGenre><abstract>Abstract: Double-stranded DNA fragments terminated at their 5′-ends by the singlestranded sequence pA-A-T-T-, generated by digestion of DNA with EcoRI restriction endonuclease, were ligated with Escherichia coli polynucleotide ligase under various conditions of temperature, concentration and time. The linear and circular products of ligation were separated by electrophoresis in agarose gel and quantitated by densitometry. The rate of ligation of (EcoRI-cleaved) simian virus (SV40) DNA at a concentration of 100 μg/ml increased from 0 °C to 5 °C to 10 °C (6-fold increase overall); raising the temperature to 15 °C did not further increase the rate of ligation. At the appropriate DNA concentrations, the predominant products of ligation are either linear concatemers that are integral multimers of the starting DNA fragment, or covalently closed circular structures of the monomeric DNA fragment. Ligating a mixture of two different length DNA fragments gives rise to all of the possible expected recombinant molecules. Linear or circular products of ligation were predicted by consideration of the total concentration of DNA termini, i, and the local concentration of one terminus in the neighborhood of the other on the same DNA molecule, j. The parameter j is a function of the length of a DNA molecule, providing this length is greater than the random coil segment of DNA. Experimentally it was found that circular structures are formed in significant amounts only under conditions when the value of j is several times greater than that of i. When j = i, equal amounts of linear and circular products would be expected, but most of the molecules were ligated into linear concatemers. No circular structure of a DNA fragment whose contour length l (6 × 10−2 μm) is smaller than the random coil segment value b (7·17 × 10−2 μm) was observed, while circular structures of the dimer of the same molecule (12 × 10−2 μm) were detected.</abstract><qualityIndicators><score>10</score><pdfWordCount>6718</pdfWordCount><pdfCharCount>37239</pdfCharCount><pdfVersion>1.3</pdfVersion><pdfPageCount>20</pdfPageCount><pdfPageSize>468 x 684 pts</pdfPageSize><refBibsNative>true</refBibsNative><abstractWordCount>310</abstractWordCount><abstractCharCount>1938</abstractCharCount><keywordCount>0</keywordCount></qualityIndicators><title>Ligation of Eco RI endonuclease-generated DNA fragments into linear and circular structures</title><pmid><json:string>169355</json:string></pmid><pii><json:string>0022-2836(75)90189-8</json:string></pii><genre><json:string>research-article</json:string></genre><host><title>Journal of Molecular Biology</title><language><json:string>unknown</json:string></language><publicationDate>1975</publicationDate><issn><json:string>0022-2836</json:string></issn><pii><json:string>S0022-2836(00)X0746-2</json:string></pii><volume>96</volume><issue>1</issue><pages><first>171</first><last>178</last></pages><genre><json:string>journal</json:string></genre></host><namedEntities><unitex><date><json:string>10s</json:string><json:string>8000</json:string><json:string>1975</json:string></date><geogName></geogName><orgName><json:string>C Apparatus Corp.</json:string></orgName><orgName_funder></orgName_funder><orgName_provider></orgName_provider><persName><json:string>Xan Francisco</json:string><json:string>U.S. Public</json:string><json:string>Ned Mantei</json:string><json:string>C. 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(1973)</json:string><json:string>Morrow et al., 1974</json:string><json:string>Old et al., 1976</json:string><json:string>Dugaiczyk et al., 1974</json:string><json:string>Wu & Taylor, 1971</json:string><json:string>MacHattie $ Thomas, 1964</json:string><json:string>Old et al., 1975</json:string><json:string>Guerry et al., 1973</json:string><json:string>Helling et al., 1974</json:string><json:string>Davidson & Szybalski, 1971</json:string><json:string>Nathans et al., 1974</json:string><json:string>Fisher & Dingman, 1971</json:string><json:string>Yoshike & F-0, 1969</json:string><json:string>Padmanabhan & Wu, 1972</json:string><json:string>Tooze, 1973</json:string><json:string>Modrich et al. (1973)</json:string><json:string>Sharp et al., 1973</json:string><json:string>Greene et al. (1974)</json:string><json:string>Boyer et al., 1973</json:string><json:string>Hirt, 1967</json:string></ref_bibl><bibl></bibl></unitex></namedEntities><ark><json:string>ark:/67375/6H6-N6XS16BC-0</json:string></ark><categories><wos><json:string>1 - science</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 - biochemistry & molecular biology</json:string></scienceMetrix><scopus><json:string>1 - Life Sciences</json:string><json:string>2 - Biochemistry, Genetics and Molecular Biology</json:string><json:string>3 - Molecular Biology</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>1975</publicationDate><copyrightDate>1975</copyrightDate><doi><json:string>10.1016/0022-2836(75)90189-8</json:string></doi><id>95F72D731B104A5926D19504CA826E84B3E41529</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-N6XS16BC-0/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-N6XS16BC-0/bundle.zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/6H6-N6XS16BC-0/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a">Ligation of Eco RI endonuclease-generated DNA fragments into linear and circular structures</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher scheme="https://scientific-publisher.data.istex.fr">ELSEVIER</publisher><availability><licence><p>elsevier</p></licence></availability><p scheme="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M"></p><date>1975</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>This investigation was supported by a U.S. Public Health Service grant (CA14026, GM14378).</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a">Ligation of Eco RI endonuclease-generated DNA fragments into linear and circular structures</title><author xml:id="author-0000"><persName><forename type="first">Achilles</forename><surname>Dugaiczyk</surname></persName><affiliation>Department of Biochemistry and Biophysics University of California School of Medicine, San Francisco Calif. 94143, U.S.A.</affiliation></author><author xml:id="author-0001"><persName><forename type="first">Herbert W.</forename><surname>Boyer</surname></persName><affiliation>Department of Microbiology University of California School of Medicine, San Francisco Calif. 94143, U.S.A.</affiliation></author><author xml:id="author-0002"><persName><forename type="first">Howard M.</forename><surname>Goodman</surname></persName><affiliation>Department of Biochemistry and Biophysics University of California School of Medicine, San Francisco Calif. 94143, U.S.A.</affiliation></author><idno type="istex">95F72D731B104A5926D19504CA826E84B3E41529</idno><idno type="ark">ark:/67375/6H6-N6XS16BC-0</idno><idno type="DOI">10.1016/0022-2836(75)90189-8</idno><idno type="PII">0022-2836(75)90189-8</idno></analytic><monogr><title level="j">Journal of Molecular Biology</title><title level="j" type="abbrev">YJMBI</title><idno type="pISSN">0022-2836</idno><idno type="PII">S0022-2836(00)X0746-2</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1975"></date><biblScope unit="volume">96</biblScope><biblScope unit="issue">1</biblScope><biblScope unit="page" from="171">171</biblScope><biblScope unit="page" to="178">178</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>1975</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>Abstract: Double-stranded DNA fragments terminated at their 5′-ends by the singlestranded sequence pA-A-T-T-, generated by digestion of DNA with EcoRI restriction endonuclease, were ligated with Escherichia coli polynucleotide ligase under various conditions of temperature, concentration and time. The linear and circular products of ligation were separated by electrophoresis in agarose gel and quantitated by densitometry. The rate of ligation of (EcoRI-cleaved) simian virus (SV40) DNA at a concentration of 100 μg/ml increased from 0 °C to 5 °C to 10 °C (6-fold increase overall); raising the temperature to 15 °C did not further increase the rate of ligation. At the appropriate DNA concentrations, the predominant products of ligation are either linear concatemers that are integral multimers of the starting DNA fragment, or covalently closed circular structures of the monomeric DNA fragment. Ligating a mixture of two different length DNA fragments gives rise to all of the possible expected recombinant molecules. Linear or circular products of ligation were predicted by consideration of the total concentration of DNA termini, i, and the local concentration of one terminus in the neighborhood of the other on the same DNA molecule, j. The parameter j is a function of the length of a DNA molecule, providing this length is greater than the random coil segment of DNA. Experimentally it was found that circular structures are formed in significant amounts only under conditions when the value of j is several times greater than that of i. When j = i, equal amounts of linear and circular products would be expected, but most of the molecules were ligated into linear concatemers. No circular structure of a DNA fragment whose contour length l (6 × 10−2 μm) is smaller than the random coil segment value b (7·17 × 10−2 μm) was observed, while circular structures of the dimer of the same molecule (12 × 10−2 μm) were detected.</p></abstract></profileDesc><revisionDesc><change when="1975">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-N6XS16BC-0/fulltext.txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Elsevier, elements deleted: 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:docType><istex:document><converted-article version="4.5.2" docsubtype="fla"><item-info><jid>YJMBI</jid><aid>75901898</aid><ce:pii>0022-2836(75)90189-8</ce:pii><ce:doi>10.1016/0022-2836(75)90189-8</ce:doi><ce:copyright type="unknown" year="1975"></ce:copyright></item-info><head><ce:article-footnote><ce:label>☆</ce:label><ce:note-para>This investigation was supported by a U.S. Public Health Service grant (CA14026, GM14378).</ce:note-para></ce:article-footnote><ce:title>Ligation of <ce:italic>Eco</ce:italic>RI endonuclease-generated DNA fragments into linear and circular structures</ce:title><ce:author-group><ce:author><ce:given-name>Achilles</ce:given-name><ce:surname>Dugaiczyk</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>1</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Herbert W.</ce:given-name><ce:surname>Boyer</ce:surname><ce:cross-ref refid="AFF2"><ce:sup>2</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Howard M.</ce:given-name><ce:surname>Goodman</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>1</ce:sup></ce:cross-ref></ce:author><ce:affiliation id="AFF1"><ce:label>1</ce:label><ce:textfn>Department of Biochemistry and Biophysics University of California School of Medicine, San Francisco Calif. 94143, U.S.A.</ce:textfn></ce:affiliation><ce:affiliation id="AFF2"><ce:label>2</ce:label><ce:textfn>Department of Microbiology University of California School of Medicine, San Francisco Calif. 94143, U.S.A.</ce:textfn></ce:affiliation></ce:author-group><ce:date-received day="20" month="1" year="1975"></ce:date-received><ce:abstract><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para>Double-stranded DNA fragments terminated at their 5′-ends by the singlestranded sequence pA-A-T-T-, generated by digestion of DNA with <ce:italic>Eco</ce:italic>RI restriction endonuclease, were ligated with <ce:italic>Escherichia coli</ce:italic> polynucleotide ligase under various conditions of temperature, concentration and time. The linear and circular products of ligation were separated by electrophoresis in agarose gel and quantitated by densitometry. The rate of ligation of (<ce:italic>Eco</ce:italic>RI-cleaved) simian virus (SV40) DNA at a concentration of 100 μg/ml increased from 0 °C to 5 °C to 10 °C (6-fold increase overall); raising the temperature to 15 °C did not further increase the rate of ligation. At the appropriate DNA concentrations, the predominant products of ligation are either linear concatemers that are integral multimers of the starting DNA fragment, or covalently closed circular structures of the monomeric DNA fragment. Ligating a mixture of two different length DNA fragments gives rise to all of the possible expected recombinant molecules.</ce:simple-para><ce:simple-para>Linear or circular products of ligation were predicted by consideration of the total concentration of DNA termini, <ce:italic>i</ce:italic>, and the local concentration of one terminus in the neighborhood of the other on the same DNA molecule, <ce:italic>j</ce:italic>. The parameter <ce:italic>j</ce:italic> is a function of the length of a DNA molecule, providing this length is greater than the random coil segment of DNA. Experimentally it was found that circular structures are formed in significant amounts only under conditions when the value of <ce:italic>j</ce:italic> is several times greater than that of <ce:italic>i</ce:italic>. When <ce:italic>j</ce:italic> = <ce:italic>i</ce:italic>, equal amounts of linear and circular products would be expected, but most of the molecules were ligated into linear concatemers. No circular structure of a DNA fragment whose contour length <ce:italic>l</ce:italic> (6 × 10<ce:sup>−2</ce:sup> μm) is smaller than the random coil segment value <ce:italic>b</ce:italic> (7·17 × 10<ce:sup>−2</ce:sup> μm) was observed, while circular structures of the dimer of the same molecule (12 × 10<ce:sup>−2</ce:sup> μm) were detected.</ce:simple-para></ce:abstract-sec></ce:abstract></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo><title>Ligation of Eco RI endonuclease-generated DNA fragments into linear and circular structures</title></titleInfo><titleInfo type="alternative" contentType="CDATA"><title>Ligation of</title></titleInfo><name type="personal"><namePart type="given">Achilles</namePart><namePart type="family">Dugaiczyk</namePart><affiliation>Department of Biochemistry and Biophysics University of California School of Medicine, San Francisco Calif. 94143, U.S.A.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Herbert W.</namePart><namePart type="family">Boyer</namePart><affiliation>Department of Microbiology University of California School of Medicine, San Francisco Calif. 94143, U.S.A.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Howard M.</namePart><namePart type="family">Goodman</namePart><affiliation>Department of Biochemistry and Biophysics University of California School of Medicine, San Francisco Calif. 94143, U.S.A.</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">1975</dateIssued><copyrightDate encoding="w3cdtf">1975</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract lang="en">Abstract: Double-stranded DNA fragments terminated at their 5′-ends by the singlestranded sequence pA-A-T-T-, generated by digestion of DNA with EcoRI restriction endonuclease, were ligated with Escherichia coli polynucleotide ligase under various conditions of temperature, concentration and time. The linear and circular products of ligation were separated by electrophoresis in agarose gel and quantitated by densitometry. The rate of ligation of (EcoRI-cleaved) simian virus (SV40) DNA at a concentration of 100 μg/ml increased from 0 °C to 5 °C to 10 °C (6-fold increase overall); raising the temperature to 15 °C did not further increase the rate of ligation. At the appropriate DNA concentrations, the predominant products of ligation are either linear concatemers that are integral multimers of the starting DNA fragment, or covalently closed circular structures of the monomeric DNA fragment. Ligating a mixture of two different length DNA fragments gives rise to all of the possible expected recombinant molecules. Linear or circular products of ligation were predicted by consideration of the total concentration of DNA termini, i, and the local concentration of one terminus in the neighborhood of the other on the same DNA molecule, j. The parameter j is a function of the length of a DNA molecule, providing this length is greater than the random coil segment of DNA. Experimentally it was found that circular structures are formed in significant amounts only under conditions when the value of j is several times greater than that of i. When j = i, equal amounts of linear and circular products would be expected, but most of the molecules were ligated into linear concatemers. No circular structure of a DNA fragment whose contour length l (6 × 10−2 μm) is smaller than the random coil segment value b (7·17 × 10−2 μm) was observed, while circular structures of the dimer of the same molecule (12 × 10−2 μm) were detected.</abstract><note>This investigation was supported by a U.S. Public Health Service grant (CA14026, GM14378).</note><relatedItem type="host"><titleInfo><title>Journal of Molecular Biology</title></titleInfo><titleInfo type="abbreviated"><title>YJMBI</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">1975</dateIssued></originInfo><identifier type="ISSN">0022-2836</identifier><identifier type="PII">S0022-2836(00)X0746-2</identifier><part><date>1975</date><detail type="volume"><number>96</number><caption>vol.</caption></detail><detail type="issue"><number>1</number><caption>no.</caption></detail><extent unit="issue-pages"><start>1</start><end>216</end></extent><extent unit="pages"><start>171</start><end>178</end></extent></part></relatedItem><identifier type="istex">95F72D731B104A5926D19504CA826E84B3E41529</identifier><identifier type="ark">ark:/67375/6H6-N6XS16BC-0</identifier><identifier type="DOI">10.1016/0022-2836(75)90189-8</identifier><identifier type="PII">0022-2836(75)90189-8</identifier><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></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-N6XS16BC-0/record.json</uri></json:item></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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