Transposon-based approaches for generating novel molecular diversity during directed evolution

Jones, Darran Dafydd ORCID: https://orcid.org/0000-0001-7709-3995, Arpino, James A. J., Baldwin, Amy Joy ORCID: https://orcid.org/0000-0002-2162-3771 and Edmundson, Matthew C. 2014. Transposon-based approaches for generating novel molecular diversity during directed evolution. Gillam, Elizabeth M. J., Copp, Janine M. and Ackerley, David, eds. Directed Evolution Library Creation: Methods and Protocols, Vol. 1179. Methods in Molecular Biology, Springer, pp. 159-172. (10.1007/978-1-4939-1053-3_11)

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Abstract

This chapter introduces a set of transposon-based methods that were developed to sample trinucleotide deletion, trinucleotide replacement, and domain insertion. Each approach has a common initial step that utilizes an engineered version of the Mu transposon called MuDel. The inherent low sequence specificity of MuDel results in its random insertion into target DNA during in vitro transposition. Removal of the transposon using a type IIS restriction endonuclease generates blunt-end random breaks at a frequency of one per target gene and the concomitant loss of 3 bp. Self-ligation or insertion of another DNA cassette results in the sampling of trinucleotide deletion or trinucleotide substitution/domain insertion, respectively.

Item Type:	Book Section
Date Type:	Publication
Status:	Published
Schools:	Biosciences
Additional Information:	PDF uploaded in accordance with publisher's policies at http://www.sherpa.ac.uk/romeo/issn/1064-3745/ (accessed 11.02.16).
Publisher:	Springer
ISBN:	9781493910526
ISSN:	1064-3745
Last Modified:	24 Nov 2024 09:30
URI:	https://orca.cardiff.ac.uk/id/eprint/64396

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