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

Gloeckner, Christian; Kranaster, Ramon; Marx, Andreas

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

Dateien

Zu diesem Dokument gibt es keine Dateien.

Datum

2009

Autor:innen

Gloeckner, Christian

Kranaster, Ramon

Marx, Andreas

Herausgeber:innen

Mahal, Lara

Romesberg, Floyd

Shah, Kavita

Shamu, Caroline

Strano, Michael S.

Thomas, Craig

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.

Fachgebiet (DDC)

540 Chemie

Schlagwörter

DNA polymerase, directed evolution, screening, PCR, primer extension

Zitieren

ISO 690

GLOECKNER, 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}
}

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