Part I. Point Mutagenesis
(i) Random Mutagenesis Methods
1. Error-Prone PCR and Effective Generation of Gene Variant Libraries for Directed Evolution
Janine N. Copp, Paulina Hanson-Manful, David F. Ackerley, and Wayne M. Patrick
2. Error-Prone Rolling Circle Amplification Greatly Simplifies Random Mutagenesis
Ryota Fujii, Motomitsu Kitaoka, and Kiyoshi Hayashi
3. Random Mutagenesis by Error-Prone Pol Plasmid Replication in Escherichia coli
David L. Alexander, Joshua Lilly, Jaime Hernandez, Jillian Romsdahl, Christopher J. Troll, and Manel Camps
4. The Sequence Saturation Mutagenesis (SeSaM) Method
Anna Joëlle Ruff, Tsvetan Kardashliev, Alexander Dennig, and Ulrich Schwaneberg
5. Generation of Effective Libraries by Neutral Drift
Miriam Kaltenbach and Nobuhiko Tokuriki
(ii) Saturation Mutagenesis Methods
6. Site Saturation Mutagenesis
Elsie M. Williams, Janine N. Copp, and David F. Ackerley
7. Iterative Saturation Mutagenesis (ISM): A Powerful Approach to Engineer Proteins by Systematically Simulating Darwinian Evolution
Carlos G. Acevedo-Rocha, Sabrina Killeand, and Manfred T. Reetz
8. Generating Targeted Libraries by the Combinatorial Incorporation of Synthetic Oligonucleotides During Gene Shuffling (ISOR)
Liat Rockah-Shmuel, Dan S Tawfik, and Moshe Goldsmith
9. Omni Change: Simultaneous Site-Saturation of up to Five Codons
Alexander Dennig, Jan Marienhagen, Anna Joelle Ruff, and Ulrich Schwaneberg
(iii) Insertional/Deletional Methods
10. Random Insertional-Deletional Strand Exchange Mutagenesis (RAISE): A Simple Method for Generating Random Insertion and Deletion Mutations
Ryota Fujii, Motomitsu Kitaoka, and Kiyoshi Hayashi
11. Transposon-Based Approaches for Generating Novel Molecular Diversity during Directed Evolution
Dafydd Jones, James A.J. Arpino, Amy J. Baldwin, and Matthew C. Edmundson
Part II. Recombinatorial Methods
(i)Homology-Dependent Methods
12. Restriction Enzyme-Mediated DNA Family Shuffling
James B.Y.H. Behrendorff, Wayne A. Johnston, and Elizabeth M. J. Gillam
13. Assembly of Designed Oligonucleotides (ADO): A Useful Tool in Synthetic Biology for Creating High Quality Combinatorial DNA Libraries
Carlos G. Acevedo-Rocha and Manfred T. Reetz
14. One-Pot, Simple Methodology for Cassette Randomization and Recombination for Focused Directed Evolution (OSCARR)
Aurelio Hidalgo Anna Schließmann, and Uwe T. Bornscheuer
15. USER Friendly DNA Recombination (USERec): Gene Library Construction Requiring Minimal Sequence Homology
Benoit Villiers and Florian Hollfelder
(ii) Homology-Independent Methods
16. ITCHY: Incremental Truncation for the Creation of Hybrid Enzymes
Wayne M. Patrick and Monica L. Gerth
17. Generating Random Circular Permutation Libraries
Stefan Lutz, Ashley B. Daugherty, Ying Yu, and Zhen Qian
Part III. Structure Guided Methods and Computational Tools for the Design and Analysis of Mutant Libraries
18. Probabilistic Methods in Directed Evolution: Library Size, Mutation Rate, and Diversity
Yuval Nov
19. The Mutagenesis Assistant P
