Abstract
This protocol describes a directed evolution method for in vitro mutagenesis and recombination of polynucleotide sequences. The staggered extension process (StEP) is essentially a modified PCR that uses highly abbreviated annealing and extension steps to generate staggered DNA fragments and promote crossover events along the full length of the template sequence(s). The resulting library of chimeric polynucleotide sequence(s) is subjected to subsequent high-throughput functional analysis. The recombination efficiency of the StEP method is comparable to that of the most widely used in vitro DNA recombination method, DNA shuffling. However, the StEP method does not require DNA fragmentation and can be carried out in a single tube. This protocol can be completed in 4–6 h.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Arnold, F.H. Combinatorial and computational challenges for biocatalyst design. Nature 409, 253–257 (2001).
Arnold, F.H. (ed.) Evolutionary Protein Design (Advances in Protein Chemistry, Vol. 55) (Academic, San Diego, USA, 2001).
Schmidt-Dannert, C. Directed evolution of single proteins, metabolic pathways, and viruses. Biochemistry 40, 13125–13136 (2001).
Stemmer, W.P. Rapid evolution of a protein in vitro by DNA shuffling. Nature 370, 389–391 (1994).
Holland, J.F. Genetic algorithms. Sci. Am. 267, 66–72 (1992).
Forrest, S. Genetic algorithms: principles of natural selection applied to computation. Science 261, 872–878 (1993).
Zhao, H., Giver, L., Shao, Z., Affholter, J.A. & Arnold, F.H. Molecular evolution by staggered extension process (StEP) in vitro recombination. Nat. Biotechnol. 16, 258–261 (1998).
Stemmer, W.P. DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc. Natl. Acad. Sci. USA 91, 10747–10751 (1994).
Zhao, H. & Arnold, F.H. Optimization of DNA shuffling for high fidelity recombination. Nucleic Acids Res. 25, 1307–1308 (1997).
Ninkovic, M., Dietrich, R., Aral, G. & Schwienhorst, A. High-fidelity in vitro recombination using a proofreading polymerase. Biotechniques 30, 530–536 (2001).
Shao, Z., Zhao, H., Giver, L. & Arnold, F.H. Random-priming in vitro recombination: an effective tool for directed evolution. Nucleic Acids Res. 26, 681–683 (1998).
Coco, W.M. et al. DNA shuffling method for generating highly recombined genes and evolved enzymes. Nat. Biotechnol. 19, 354–359 (2001).
Gibbs, M.D., Nevalainen, K.M. & Bergquist, P.L. Degenerate oligonucleotide gene shuffling (DOGS): a method for enhancing the frequency of recombination with family shuffling. Gene 271, 13–20 (2001).
Coco, W.M. et al. Growth factor engineering by degenerate homoduplex gene family recombination. Nat. Biotechnol. 20, 1246–1250 (2002).
Ness, J.E. et al. Synthetic shuffling expands functional protein diversity by allowing amino acids to recombine independently. Nat. Biotechnol. 20, 1251–1255 (2002).
Ostermeier, M., Shim, J.H. & Benkovic, S.J. A combinatorial approach to hybrid enzymes independent of DNA homology. Nat. Biotechnol. 17, 1205–1209 (1999).
Sieber, V., Martinez, C.A. & Arnold, F.H. Libraries of hybrid proteins from distantly related sequences. Nat. Biotechnol. 19, 456–460 (2001).
Bittker, J.A., Le, B.V. & Liu, D.R. Nucleic acid evolution and minimization by nonhomologous random recombination. Nat. Biotechnol. 20, 1024–1029 (2002).
Volkov, A.A., Shao, Z. & Arnold, F.H. Recombination and chimeragenesis by in vitro heteroduplex formation and in vivo repair. Nucleic Acids Res. 27, e18 (1999).
Saiki, R.K. et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA-polymerase. Science 239, 487–491 (1988).
Meyerhans, A., Vartanian, J.P. & Wainhobson, S. DNA recombination during PCR. Nucleic Acids Res. 18, 1687–1691 (1990).
Hu, W.S., Bowman, E.H., Delviks, K.A. & Pathak, V.K. Homologous recombination occurs in a distinct retroviral subpopulation and exhibits high negative interference. J. Virol. 71, 6028–6036 (1997).
Zhao, H. Staggered extension process in vitro DNA recombination. Methods Enzymol. 388, 42–49 (2004).
Aguinaldo, A.M. & Arnold, F. Staggered extension process (StEP) in vitro recombination. Methods Mol. Biol. 192, 235–239 (2002).
Volkov, A.A. & Arnold, F.H. Methods for in vitro DNA recombination and random chimeragenesis. Methods Enzymol. 328, 447–456 (2000).
Zhao, H. & Arnold, F.H. Directed evolution converts subtilisin E into a functional equivalent of thermitase. Protein Eng. 12, 47–53 (1999).
Glieder, A., Farinas, E.T. & Arnold, F.H. Laboratory evolution of a soluble, self-sufficient, highly active alkane hydroxylase. Nat. Biotechnol. 20, 1135–1139 (2002).
Ghadessy, F.J. et al. Generic expansion of the substrate spectrum of a DNA polymerase by directed evolution. Nat. Biotechnol. 22, 755–759 (2004).
Maheshri, N., Koerber, J.T., Kaspar, B.K. & Schaffer, D.V. Directed evolution of adeno-associated virus yields enhanced gene delivery vectors. Nat. Biotechnol. 24, 198–204 (2006).
Dion, M. et al. Modulation of the regioselectivity of a Bacillus alpha-galactosidase by directed evolution. Glycoconj. J. 18, 215–223 (2001).
He, M., Yang, Z.Y., Nie, Y.F., Wang, J. & Xu, P.L. A new type of class I bacterial 5-enopyruvylshikimate-3-phosphate synthase mutants with enhanced tolerance to glyphosate. Biochim. Biophys. Acta Gen. Subjects 1568, 1–6 (2001).
Murashima, K., Kosugi, A. & Doi, R.H. Thermostabilization of cellulosomal endoglucanase EngB from Clostridium cellulovorans by in vitro DNA recombination with non-cellulosomal endoglucanase EngD. Mol. Microbiol. 45, 617–626 (2002).
Bulter, T. et al. Functional expression of a fungal laccase in Saccharomyces cerevisiae by directed evolution. Appl. Environ. Microbiol. 69, 987–995 (2003).
Wu, Z.L., Podust, L.M. & Guengerich, F.P. Expansion of substrate specificity of cytochrome P450 2A6 by random and site-directed mutagenesis. J. Biol. Chem. 280, 41090–41100 (2005).
Miyazaki, K. et al. Thermal stabilization of Bacillus subtilis family-11 xylanase by directed evolution. J. Biol. Chem. 281, 10236–10242 (2006).
Sheedy, C., Yau, K.Y.F., Hirama, T., MacKenzie, C.R. & Hall, J.C. Selection, characterization, and CDR shuffling of naive llama single-domain antibodies selected against auxin and their cross-reactivity with auxinic herbicides from four chemical families. J. Agric. Food Chem. 54, 3668–3678 (2006).
Innis, M.A., Myambo, K.B., Gelfand, D.H. & Brow, M.A. DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc. Natl. Acad. Sci. USA 85, 9436–9440 (1988).
Judo, M.S., Wedel, A.B. & Wilson, C. Stimulation and suppression of PCR-mediated recombination. Nucleic Acids Res. 26, 1819–1825 (1998).
Acknowledgements
We thank the Department of Defense (N000140210725), National Science Foundation (BES-0348107), National Institutes of Health, and DuPont for supporting our work on development and applications of new directed evolution tools for protein science and engineering and metabolic engineering.
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Rights and permissions
About this article
Cite this article
Zhao, H., Zha, W. In vitro 'sexual' evolution through the PCR-based staggered extension process (StEP). Nat Protoc 1, 1865–1871 (2006). https://doi.org/10.1038/nprot.2006.309
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2006.309
This article is cited by
-
Improving the activity and expression level of a phthalate-degrading enzyme by a combination of mutagenesis strategies and strong promoter replacement
Environmental Science and Pollution Research (2023)
-
Chemically stable fluorescent proteins for advanced microscopy
Nature Methods (2022)
-
Directed evolution of the B. subtilis nitroreductase YfkO improves activation of the PET-capable probe SN33623 and CB1954 prodrug
Biotechnology Letters (2021)
-
Optimizing Human Epidermal Growth Factor for its Endurance and Specificity Via Directed Evolution: Functional Importance of Leucine at Position 8
International Journal of Peptide Research and Therapeutics (2020)
-
Ribosome Display: A Potent Display Technology used for Selecting and Evolving Specific Binders with Desired Properties
Molecular Biotechnology (2019)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.