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

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,

SUBMITTER: Stein V 

PROVIDER: S-EPMC2764453 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature