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Published online 10 February 2004

Nucleic Acids Research, 2004, Vol. 32, No. 3 e26
© 2004 Oxford University Press

Sequence saturation mutagenesis (SeSaM): a novel method for directed evolution

Tuck Seng Wong, Kang Lan Tee, Berhard Hauer1 and Ulrich Schwaneberg*

International University Bremen (IUB), Campus Ring 1, 28759 Bremen, Germany 1 Fine Chemicals and Biocatalysis Research, GVF/D-B009, 67056 Ludwigshafen, Germany

*To whom correspondence should be addressed. Tel: +49 421 200 3543; Fax: +49 421 200 3229; Email: u.schwaneberg{at}iu-bremen.de

Sequence saturation mutagenesis (SeSaM) is a conceptually novel and practically simple method that truly randomizes a target sequence at every single nucleotide position. A SeSaM experiment can be accomplished within 2–3 days and comprises four steps: generating a pool of DNA fragments with random length, ‘tailing’ the DNA fragments with universal base using terminal transferase at 3'-termini, elongating DNA fragments in a PCR to the full-length genes using a single-stranded template and replacing the universal bases by standard nucleotides. Random mutations are created at universal sites due to the promiscuous base-pairing property of universal bases. Using enhanced green fluorescence protein as the model system and deoxyinosine as the universal base, we proved by sequencing 100 genes the concept of the SeSaM method and achieved a random distribution of mutations with the mutational bias expected for deoxyinosine.


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