Publikation: Directed evolution of DNA Polymerases : construction and screening of DNA Polymerase Mutant Libraries
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Datum
2009
Autor:innen
Herausgeber:innen
Mahal, Lara
Romesberg, Floyd
Shah, Kavita
Shamu, Caroline
Strano, Michael S.
Thomas, Craig
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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.
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Fachgebiet (DDC)
540 Chemie
Schlagwörter
DNA polymerase, directed evolution, screening, PCR, primer extension
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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.ch090183BibTex
@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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<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>
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