Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates


Saturation mutagenesis constitutes a powerful method in the directed evolution of enzymes. Traditional protocols of whole plasmid amplification such as Stratagene’s QuikChange™ sometimes fail when the templates are difficult to amplify. In order to overcome such restrictions, we have devised a simple two-primer, two-stage polymerase chain reaction (PCR) method which constitutes an improvement over existing protocols. In the first stage of the PCR, both the mutagenic primer and the antiprimer that are not complementary anneal to the template. In the second stage, the amplified sequence is used as a megaprimer. Sites composed of one or more residues can be randomized in a single PCR reaction, irrespective of their location in the gene sequence.The method has been applied to several enzymes successfully, including P450-BM3 from Bacillus megaterium, the lipases from Pseudomonas aeruginosa and Candida antarctica and the epoxide hydrolase from Aspergillus niger. Here, we show that megaprimer size as well as the direction and design of the antiprimer are determining factors in the amplification of the plasmid. Comparison of the results with the performances of previous protocols reveals the efficiency of the improved method.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5


  1. Arndt KM, Müller KM (2007) Protein engineering protocols (Methods in molecular biology), vol. 352. Humana, Totowa

  2. Arnold FH, Georgiou G (2003a) Directed enzyme evolution: Screening and selection methods (Methods in molecular biology), vol. 230. Humana, Totowa

  3. Arnold FH, Georgiou G (2003b) Directed evolution library creation: methods and protocols. In: Methods in molecular biology, vol. 231. Humana, Totowa, p 75-84

  4. Bartsch S, Kourist R, Bornscheuer UT (2008) Complete inversion of enantioselectivity towards acetylated tertiary alcohols by a double mutant of a Bacillus subtilis esterase. Angew Chem Int Ed 47:1508–1511 Angew Chem 120:1531−1534

  5. Bershtein S, Tawfik DS (2008) Advances in laboratory evolution of enzymes. Curr Opin Chem Biol 12:151–158

  6. Brakmann S, Schwienhorst A (2004) Evolutionary methods in biotechnology: clever tricks for directed evolution. Wiley, Weinheim

  7. Cadwell RC, Joyce GF (1992) Randomization of genes by PCR mutagenesis. PCR Methods Appl 2:28–33

  8. Cedrone F, Niel S, Roca S, Bhatnagar T, Ait-Abdelkader N, Torre C, Krumm H, Maichele A, Reetz MT, Baratti JC (2003) Directed evolution of the epoxide hydrolase from Aspergillus niger. Biocatal Biotransform 21:357–364

  9. Dominy CN, Andrews DW (2003) Site-directed mutagenesis by inverse PCR. In: Casali N, Preston A (eds) Methods in molecular biology, vol. 235. Humana, Totowa, pp 209–223

  10. Fox RJ, Huisman GW (2008) Enzyme optimization: moving from blind evolution to statistical exploration of sequence-function space. Trends Biotechnol 26:132–138

  11. Fujii R, Nakagawa Y, Hiratake J, Sogabe A, Sakata K (2005) Directed evolution of Pseudomonas aeruginosa lipase for improved amide-hydrolyzing activity. Prot Eng Des Sel 18:93–101

  12. Georgescu R, Bandara G, Sun L (2003) Saturation mutagenesis. In: Arnold FH, Georgiou G (eds) Directed evolution library creation. vol. 231. Humana, Totowa, pp 75–83

  13. Hibbert EG, Baganz F, Hailes HC, Ward JM, Lye GJ, Woodley JM, Dalby PA (2005) Directed evolution of biocatalytic processes. Biomol Eng 22:11–19

  14. Hogrefe HH, Cline J, Youngblood GL, Allen RM (2002) Creating randomized amino acid libraries with the QuikChange® multi site-directed mutagenesis kit. BioTechniques 33:1158–1165

  15. Jaeger K-E, Schneidinger B, Rosenau F, Werner M, Lang D, Dijkstra BW, Schimossek K, Zonta A, Reetz MT (1997) Bacterial lipases for biotechnological applications. J Mol Catal B: Enzym 3:3–12

  16. Kirsch RD, Joly E (1998) An improved PCR-mutagenesis strategy for two-site mutagenesis or sequence swapping between related genes. Nucleic Acids Res 26:1848–1850

  17. Leung DW, Chen E, Goeddel DV (1989) A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique (Philadelphia) 1:11–15

  18. Li H-M, Mei L-H, Urlacher VB, Schmid RD (2008) Cytochrome P450 BM-3 evolved by random and saturation mutagenesis as an effective indole-hydroxylating catalyst. Appl Biochem Biotechnol 144:27–36

  19. Liang L, Zhang J, Lin Z (2007) Altering coenzyme specificity of Pichia stipitis xylose reductase by the semi-rational approach CASTing. Microb Cell Fact 6:36

  20. Liebeton K, Zonta A, Schimossek K, Nardini M, Lang D, Dijkstra BW, Reetz MT, Jaeger K-E (2000) Directed evolution of an enantioselective lipase. Chem Biol 7:709–718

  21. Liebeton K, Zacharias A, Jaeger K-E (2001) Disulfide bond in Pseudomonas aeruginosa lipase stabilizes the structure but is not required for interaction with its foldase. J Bacteriol 183:597–603

  22. Lutz S, Patrick WM (2004) Novel methods for directed evolution of enzymes: quality, not quantity. Curr Opin Biotechnol 15:291–297

  23. Miyazaki K, Takenouchi M (2002) Creating random mutagenesis libraries using megaprimer PCR of whole plasmid. BioTechniques 33:1033–1038

  24. Narhi LO, Fulco AJ (1982) Phenobarbital induction of a soluble cytochrome P-450-dependent fatty acid mono-oxygenase in Bacillus megaterium. J Biol Chem 257:2147–2150

  25. Peters MW, Meinhold P, Glieder A, Arnold FH (2003) Regio- and enantioselective alkane hydroxylation with engineered cytochromes P450 BM-3. J Am Chem Soc 125:13442–13450

  26. Reetz MT (2004) Controlling the enantioselectivity of enzymes by directed evolution: Practical and theoretical ramifications. Proc Natl Acad Sci U S A 101:5716–5722

  27. Reetz MT (2006) Directed evolution of enantioselective enzymes as catalysts for organic synthesis. In: Gates BC, Knözinger H (eds) Advances in catalysis. vol. 49. Elsevier, San Diego, pp 1–69

  28. Reetz MT, Carballeira JD (2007) Iterative Saturation Mutagenesis (ISM) for rapid directed evolution of functional enzymes. Nat Protoc 2:891–903

  29. Reetz MT, Wilensek S, Zha D, Jaeger K-E (2001) Directed evolution of an enantioselective enzyme through combinatorial multiple cassette mutagenesis. Angew Chem Int Ed 40:3589–3591 Angew Chem 113:3701–3703

  30. Reetz MT, Carballeira JD, Peyralans JJ-P, Höbenreich H, Maichele A, Vogel A (2006a) Expanding the substrate scope of enzymes: Combining mutations obtained by CASTing. Chem-Eur J 12:6031–6038

  31. Reetz MT, Carballeira JD, Vogel A (2006b) Iterative saturation mutagenesis on the basis of B Factors as a strategy for increasing protein thermostability. Angew Chem Int Ed 45:7745–7751 Angew Chem 118:7909–7915

  32. Reetz MT, Wang L-W, Bocola M (2006c) Directed evolution of enantioselective enzymes: Iterative cycles of CASTing for probing protein-sequence space. Angew Chem Int Ed 45:1236–1241 Erratum 2494, Angew Chem 118:1258–1263; Erratum 2556

  33. Reetz MT, Puls M, Carballeira JD, Vogel A, Jaeger K-E, Eggert T, Thiel W, Bocola M, Otte N (2007) Learning from directed evolution: Further lessons from theoretical investigations into cooperative mutations in lipase enantioselectivity. ChemBioChem 8:106–112

  34. Reymond J-L (2005) Enzyme assays—high-throughput screening, genetic selection and fingerprinting. Wiley, Weinheim

  35. Rubin-Pitel SB, Zhao H (2006) Recent advances in biocatalysis by directed enzyme evolution. Comb Chem High Throughput Screening 9:247–257

  36. Sarkar G, Sommer SS (1990) The “megaprimer” method of site-directed mutagenesis. BioTechniques 8:404–407

  37. Stemmer WPC (1994) Rapid evolution of a protein in vitro by DNA shuffling. Nature (London, UK) 370:389–391

  38. Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warrener P, Hickey MJ, Brinkmann FSL, Hufnagle WO, Kowalik DJ, Lagrou M, Garber RL, Goltry L, Tolentino E, Westbrock-Wadman S, Yuan Y, Brody LL, Coulter SN, Folger KR, Kas A, Larbig K, Lim R, Smith K, Spencer D, Wong GK-S, Wu Z, Paulsen IT, Reizer J, Saier MH, Hancock REW, Lory S, Olson MV (2000) Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature (London, UK) 406:959–964

  39. Tee KL, Schwaneberg U (2007) Directed evolution of oxygenases: Screening system, success stories and challenges. Comb Chem High Throughput Screening 10:197–217

  40. Tseng W-C, Lin J-W, Wei T-Y, Fang T-Y (2008) A novel megaprimed and ligase-free, PCR-based, site-directed mutagenesis method. Anal Biochem 375:376–378

  41. Wong TS, Arnold FH, Schwaneberg U (2008) Laboratory evolution of cytochrome P450 BM-3 monooxygenase for organic cosolvents. Biotechnol Bioeng 85:351–358

  42. Zheng L, Baumann U, Reymond J-L (2004) An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucleic Acids Res 32:e115

Download references


This research was supported by the German–Israeli Project Cooperation (DIP), the Deutsche Forschungsgemeinschaft (Schwerpunkt 1170; “Directed Evolution to Optimize and Understand Molecular Biocatalysis”; Project RE 359/13-1) and the Fonds der Chemischen Industrie.

Open Access

This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

Author information

Correspondence to Manfred T. Reetz.

Additional information

Joaquin Sanchis, Layla Fernández, and J. Daniel Carballeira contributed equally.

Rights and permissions

Open Access This is an open access article distributed under the terms of the Creative Commons Attribution Noncommercial License (https://creativecommons.org/licenses/by-nc/2.0), which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

Reprints and Permissions

About this article

Cite this article

Sanchis, J., Fernández, L., Carballeira, J.D. et al. Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates. Appl Microbiol Biotechnol 81, 387–397 (2008). https://doi.org/10.1007/s00253-008-1678-9

Download citation


  • Directed evolution
  • Saturation mutagenesis
  • PCR
  • Megaprimer
  • Antiprimer
  • Difficult-to-amplify templates