Methods of genetic diversity creation and functional display for directed evolution experiments

11 TIIVISTELMA 12 1 INTRODUCTION 13 2 REVIEW OF THE LITERATURE 15 2.1 Methods for gene variant library construction 15 2.1.1 Random mutagenesis 15 2.1.1.1 Time 15 2.1.1.2 Mutator strains 15 2.1.1.3 Mutagens 16 2.1.1.4 Error-prone reactions with thermophilic enzymes ... 19 2.1.1.5 Error-prone reactions with mesophilic enzymes ...... 23 2.1.1.6 Transposons 24 2.1.1.7 Choosing the method 25 2.1.2 Oligonucleotide-directed mutagenesis 26 2.1.2.1 Focused random mutagenesis and doped oligonucleotides 26 2.1.2.2 Oligonucleotides with randomized codons (NNN/NNK/NDT/KMT) 27 2.1.2.3 Defined primer pools, TRIM and beyond 29 2.1.2.4 General considerations of DNA incorporation ......... 32 2.1.2.5 Construction of highly diverse libraries 33 2.1.2.6 Construction of small diversity libraries 37 2.1.3 Recombination 38 2.1.3.1 Aims and applications 38 2.1.3.2 In vivo 39 2.1.3.3 In vitro 40 2.2 Methods for display and selection 42 2.2.1 Geno-phenotype linkage and selection 42 2.2.2 High-throughput screening 43 2.2.3 Phage display 44 2.2.4 Cell surface display 46 2.2.5 Ribosome display and related instant links 48

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