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. 2004 Feb 10;32(3):e26.
doi: 10.1093/nar/gnh028.

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

Tuck Seng Wong  1 , Kang Lan Tee, Berhard Hauer, Ulrich Schwaneberg
Affiliations

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

Tuck Seng Wong et al. Nucleic Acids Res. .

Abstract

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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Figures

Figure 1
Figure 1
Scheme of the SeSaM method that comprises four steps. Step 1: creating a pool of DNA fragments with a random size distribution. Step 2: enzymic elongation of the DNA fragments with a universal base. Step 3: full-length gene synthesis. Step 4: universal base replacement by standard nucleotides.
Figure 2
Figure 2
Step 1 of SeSaM: creating a DNA fragment pool with length distribution. (A) scheme of Step 1. (B) Gel pictures: PCR products before (left lane) and after (right lane) iodine cleavage. The PCRs were performed in the presence of the stated dATPαS concentration. Bottom gel picture: DNA fragment size distribution after DNA melting and purification of the cleaved PCR products.
Figure 2
Figure 2
Step 1 of SeSaM: creating a DNA fragment pool with length distribution. (A) scheme of Step 1. (B) Gel pictures: PCR products before (left lane) and after (right lane) iodine cleavage. The PCRs were performed in the presence of the stated dATPαS concentration. Bottom gel picture: DNA fragment size distribution after DNA melting and purification of the cleaved PCR products.
Figure 3
Figure 3
Step 2 of SeSaM: enzymic elongation of DNA fragments with the universal base deoxyinosine.
Figure 4
Figure 4
Step 3 of SeSaM: full-length gene synthesis by a PCR employing a single-stranded template and a reverse primer that amplifies the newly synthesized strand.
Figure 5
Figure 5
Step 4 of SeSaM: the universal base deoxyinosine is replaced by one of the four standard nucleotides in a PCR.
Figure 6
Figure 6
Mutational spectra of the SeSaM method employing dATPαS and deoxyinosine: In, insertion; De, deletion.
Figure 7
Figure 7
Distribution of the mutations generated by the SeSaM method.
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References

    1. Brakmann S. (2001) Discovery of superior enzymes by directed molecular evolution. Chem. Biochem., 2, 865–871. - PubMed
    1. Cadwell R.C. and Joyce,G.F. (1992) Randomization of genes by PCR mutagenesis. PCR Methods Appl., 2, 28–33. - PubMed
    1. Cadwell R.C. and Joyce,G.F. (1994) Mutagenic PCR. PCR Methods Appl., 3, 136–140. - PubMed
    1. Lin-Goerke J.L., Robbins,D.J. and Burczak,J.D. (1997) PCR-based random mutagenesis using manganese and reduced dNTP concentration. Biotechniques, 23, 409–412. - PubMed
    1. Zaccolo M., Williams,D.M., Brown,D.M. and Gherardi,E. (1996) An approach to random mutagenesis of DNA using mixtures of triphosphate derivatives of nucleoside analogues. J. Mol. Biol., 255, 589–603. - PubMed

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