Abstract
Massive Mutagenesis® is a proprietary library creation method that enables the fast generation of high-quality genetic libraries. Starting from a single gene on a plasmid and hundreds to thousands of oligonucleotides, a one-step single-strand circular amplification method creates random combinations of several site-directed substitutions, insertions, or deletions. Libraries of up to a billion such variants have been routinely generated. Sequencing those variants demonstrated lower biases than alternative approaches such as error-prone PCR. Screening and selecting them has yielded improved biocatalysts, therapeutic proteins, and antibodies.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Delcourt M, Blesa S (2000) Method for massive directed mutagenesis Patents EP1311670B1 and US7202086B2
Sylvestre J, et al (In Press) Modern biopharmaceuticals In: Joerg Knaeblein (ed) Recent success stories. Wiley and Sons, Weinheim
Sylvestre J, Delcourt M (2004) Method of massive mutagenesis. Patent applications EP1544296 and US2005153343
Li F et al (2002) Site-directed mutagenesis facilitated by DpnI selection on hemimethylated DNA. Methods Mol Biol 182:19–27
Randolph J et al (2008) Codon-based mutagenesis using trimer phosphoramidites. Nucleic Acids Symp Ser 52:479
Saboulard D et al (2005) High-throughput mutagenesis using oligonucleotides synthesized on DNA chips. Biotechniques 39(3):363–68
Richmond KE et al (2004) Amplification and assembly of chip-eluted DNA (AACED): a method for high-throughput gene synthesis. Nucleic Acids Research 32(17):5011–5018
Laffly E et al (2008) Improvement of an antibody neutralizing the anthrax toxin by simultaneous mutagenesis of Its six hypervariable loops. J Mol Biol 378:1094–1103
Chautard H et al (2007) An activity-independent selection system of thermostable protein variants. Nat Methods 4:919–921
Acknowledgments
Massive Mutagenesis® is a patented technology of Biométhodes, an industrial biotechnology company based near Paris, France. Please contact Biométhodes regarding licensing conditions and partnership opportunities (www.biomethodes.com). The author thanks Ida Swarczewskaja for conceiving and drawing the figure.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Sylvestre, J. (2010). Massive Mutagenesis®: High-Throughput Combinatorial Site-Directed Mutagenesis. In: Braman, J. (eds) In Vitro Mutagenesis Protocols. Methods in Molecular Biology, vol 634. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-652-8_17
Download citation
DOI: https://doi.org/10.1007/978-1-60761-652-8_17
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-651-1
Online ISBN: 978-1-60761-652-8
eBook Packages: Springer Protocols