Directed evolution of DNA Polymerases : construction and screening of DNA Polymerase Mutant Libraries

Lade...
Vorschaubild
Dateien
Zu diesem Dokument gibt es keine Dateien.
Datum
2009
Autor:innen
Gloeckner, Christian
Herausgeber:innen
Mahal, Lara
Romesberg, Floyd
Shah, Kavita
Shamu, Caroline
Strano, Michael S.
Thomas, Craig
Kontakt
ISSN der Zeitschrift
Electronic ISSN
ISBN
Bibliografische Daten
Verlag
Schriftenreihe
Auflagebezeichnung
ArXiv-ID
Internationale Patentnummer
Angaben zur Forschungsförderung
Projekt
Open Access-Veröffentlichung
Sammlungen
Core Facility der Universität Konstanz
Gesperrt bis
Titel in einer weiteren Sprache
Forschungsvorhaben
Organisationseinheiten
Zeitschriftenheft
Publikationstyp
Zeitschriftenartikel
Publikationsstatus
Published
Erschienen in
Current Protocols in Chemical Biology. 2009, 2(2), pp. 89-109. Available under: doi: 10.1002/9780470559277.ch090183
Zusammenfassung

The protocols in this article describe the construction of a mutant DNA polymerase library using error-prone PCR (epPCR) as a method for gene randomization, followed by screening of the library using two different approaches. The examples described use an N-terminally truncated form of the thermostable DNA polymerase I of Thermus aquaticus (Taq DNA polymerase), namely Klentaq (KTQ), and protocols are included for the identiÞcation of variants with (1) increased DNA lesion-bypass ability and (2) enhanced selectivity for DNA match/mismatch recognition. The screening assays are based on double-stranded DNA detection (using SYBR Green I) which can be carried out using standard laboratory equipment. The described assays are designed for use in a 384-well plate format to increase screening throughput and reduce material costs. For improved accuracy and ease of liquid handling, the use of an automated liquid handling device is recommended.

Zusammenfassung in einer weiteren Sprache
Fachgebiet (DDC)
540 Chemie
Schlagwörter
DNA polymerase, directed evolution, screening, PCR, primer extension
Konferenz
Rezension
undefined / . - undefined, undefined
Zitieren
ISO 690GLOECKNER, Christian, Ramon KRANASTER, Andreas MARX, MAHAL, Lara, ed., Floyd ROMESBERG, ed., Kavita SHAH, ed., Caroline SHAMU, ed., Michael S. STRANO, ed., Craig THOMAS, ed., 2009. Directed evolution of DNA Polymerases : construction and screening of DNA Polymerase Mutant Libraries. In: Current Protocols in Chemical Biology. 2009, 2(2), pp. 89-109. Available under: doi: 10.1002/9780470559277.ch090183
BibTex
@article{Gloeckner2009Direc-12600,
  year={2009},
  doi={10.1002/9780470559277.ch090183},
  title={Directed evolution of DNA Polymerases : construction and screening of DNA Polymerase Mutant Libraries},
  number={2},
  volume={2},
  journal={Current Protocols in Chemical Biology},
  pages={89--109},
  author={Gloeckner, Christian and Kranaster, Ramon and Marx, Andreas}
}
RDF
<rdf:RDF
    xmlns:dcterms="http://purl.org/dc/terms/"
    xmlns:dc="http://purl.org/dc/elements/1.1/"
    xmlns:rdf="http://www.w3.org/1999/02/22-rdf-syntax-ns#"
    xmlns:bibo="http://purl.org/ontology/bibo/"
    xmlns:dspace="http://digital-repositories.org/ontologies/dspace/0.1.0#"
    xmlns:foaf="http://xmlns.com/foaf/0.1/"
    xmlns:void="http://rdfs.org/ns/void#"
    xmlns:xsd="http://www.w3.org/2001/XMLSchema#" > 
  <rdf:Description rdf:about="https://kops.uni-konstanz.de/server/rdf/resource/123456789/12600">
    <dc:date rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2011-06-16T08:43:13Z</dc:date>
    <dc:creator>Gloeckner, Christian</dc:creator>
    <dspace:isPartOfCollection rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/29"/>
    <dc:contributor>Gloeckner, Christian</dc:contributor>
    <dcterms:available rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2011-06-16T08:43:13Z</dcterms:available>
    <dc:contributor>Romesberg, Floyd</dc:contributor>
    <dc:contributor>Mahal, Lara</dc:contributor>
    <dc:contributor>Kranaster, Ramon</dc:contributor>
    <dc:rights>terms-of-use</dc:rights>
    <dcterms:title>Directed evolution of DNA Polymerases : construction and screening of DNA Polymerase Mutant Libraries</dcterms:title>
    <dc:creator>Kranaster, Ramon</dc:creator>
    <dcterms:rights rdf:resource="https://rightsstatements.org/page/InC/1.0/"/>
    <dcterms:issued>2009</dcterms:issued>
    <void:sparqlEndpoint rdf:resource="http://localhost/fuseki/dspace/sparql"/>
    <bibo:uri rdf:resource="http://kops.uni-konstanz.de/handle/123456789/12600"/>
    <dcterms:isPartOf rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/29"/>
    <dc:contributor>Shah, Kavita</dc:contributor>
    <dcterms:abstract xml:lang="eng">The protocols in this article describe the construction of a mutant DNA polymerase library using error-prone PCR (epPCR) as a method for gene randomization, followed by screening of the library using two different approaches. The examples described use an N-terminally truncated form of the thermostable DNA polymerase I of Thermus aquaticus (Taq DNA polymerase), namely Klentaq (KTQ), and protocols are included for the identiÞcation of variants with (1) increased DNA lesion-bypass ability and (2) enhanced selectivity for DNA match/mismatch recognition. The screening assays are based on double-stranded DNA detection (using SYBR Green I) which can be carried out using standard laboratory equipment. The described assays are designed for use in a 384-well plate format to increase screening throughput and reduce material costs. For improved accuracy and ease of liquid handling, the use of an automated liquid handling device is recommended.</dcterms:abstract>
    <dc:contributor>Marx, Andreas</dc:contributor>
    <dc:contributor>Strano, Michael S.</dc:contributor>
    <dc:creator>Marx, Andreas</dc:creator>
    <foaf:homepage rdf:resource="http://localhost:8080/"/>
    <dc:contributor>Thomas, Craig</dc:contributor>
    <dc:language>eng</dc:language>
    <dc:contributor>Shamu, Caroline</dc:contributor>
  </rdf:Description>
</rdf:RDF>
Interner Vermerk
xmlui.Submission.submit.DescribeStep.inputForms.label.kops_note_fromSubmitter
Kontakt
URL der Originalveröffentl.
Prüfdatum der URL
Prüfungsdatum der Dissertation
Finanzierungsart
Kommentar zur Publikation
Allianzlizenz
Corresponding Authors der Uni Konstanz vorhanden
Internationale Co-Autor:innen
Universitätsbibliographie
Ja
Begutachtet
Diese Publikation teilen