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An efficient method to assemble linear DNA templates for in vitro screening and selection systems.

Stein, Viktor and Hollfelder, Florian (2009):
An efficient method to assemble linear DNA templates for in vitro screening and selection systems.
In: Nucleic acids research, pp. e122, 37, (18), ISSN 1362-4962, [Article]

Abstract

A method is presented to assemble a gene of interest into a linear DNA template with all the components necessary for in vitro transcription and translation in approximately 90 min. Assembly is achieved using a coupled uracil excision-ligation strategy based on USER Enzyme and T4 DNA ligase, which allows the simultaneous and seamless assembly of three different PCR products. The method is suitable for screening and selection systems of very high throughput as up to 10(11) molecules can be efficiently assembled and purified in reaction volumes of 100 microl. The method is exemplified with the gene coding for a mutant version of O(6)-alkylguanine alkyltransferase, which is efficiently assembled with an N-terminal peptide tag and its 5'- and 3'-untranslated regions that include a T7 promoter, ribosome binding site and T7 terminator. The utility of the method is further corroborated by assembling error-prone PCR libraries and regenerating templates following model affinity selections. This fast and robust method should find widespread application in directed evolution for the assembly of gene libraries and the regeneration of linear DNA templates between successive screening and selection cycles.

Item Type: Article
Erschienen: 2009
Creators: Stein, Viktor and Hollfelder, Florian
Title: An efficient method to assemble linear DNA templates for in vitro screening and selection systems.
Language: English
Abstract:

A method is presented to assemble a gene of interest into a linear DNA template with all the components necessary for in vitro transcription and translation in approximately 90 min. Assembly is achieved using a coupled uracil excision-ligation strategy based on USER Enzyme and T4 DNA ligase, which allows the simultaneous and seamless assembly of three different PCR products. The method is suitable for screening and selection systems of very high throughput as up to 10(11) molecules can be efficiently assembled and purified in reaction volumes of 100 microl. The method is exemplified with the gene coding for a mutant version of O(6)-alkylguanine alkyltransferase, which is efficiently assembled with an N-terminal peptide tag and its 5'- and 3'-untranslated regions that include a T7 promoter, ribosome binding site and T7 terminator. The utility of the method is further corroborated by assembling error-prone PCR libraries and regenerating templates following model affinity selections. This fast and robust method should find widespread application in directed evolution for the assembly of gene libraries and the regeneration of linear DNA templates between successive screening and selection cycles.

Journal or Publication Title: Nucleic acids research
Volume: 37
Number: 18
Divisions: 10 Department of Biology
10 Department of Biology > Protein Engineering of Ion Conducting Nanopores
Date Deposited: 14 Nov 2016 11:43
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