Directe d Mutagenesis A Practical Approac h M. J. McPHERSON
1. Mutagenesis facilitated by the removal or introduction of unique restriction sites 1 P. Carte r 1. Introduction to site-directed mutagenesis 1 Mutagenesis using mismatched oligonucleotides 1 Factors reducing yield 2 Strategies to enhance mutant yield 4 Phagemid vectors 5 Design of mutagenic oligonucleotide primers 6 Choice of endonuclease for restriction-selection and restriction - purification 8 Methods included in this chapter 9 2. Oligonucleotide-directed mutagenesis facilitated b y using repair deficient host strains and restrictionselection or restriction-purification 9 Enzymes 9 Other reagents 9 Bacterial and phage strains 1 0 Media 1 0 Preparation of single-stranded phagemid DNA 1 0 Construction of site-directed mutations 1 1 3. Trouble-shooting procedures 1 9 Likely origin of clones other than target mutant 1 9 Checks on the efficiency of mutagenesis 20 Specificity of priming of mutagenic oligonucleotide 2 1 Purity of mutagenic oligonucleotide 2 1 Extent of phosphorylation of mutant oligonucleotide 2 1 4. Advantages and limitations of restriction-selection and restriction-purification 2 2 Acknowledgements 2 3 References 23
2. Site-directed in vitro mutagenesis usin g uracil-containing DNA and phagemi d vectors 27 P. D. Yuckenberg, F. Witney, J. Geisselsoder, and J. McClary 1. Introduction 27 2. Mutagenesis procedures 3 1 Bacteriology 3 1 Introduction of phagemid into E. coli CJ236 3 2 Isolation of uracil-containing phagemid particles 3 6 Extraction of phagemid DNA 3 8 Synthesis of the mutagenic strand 3 9 Use of T4 gene 32 protein 45 Gel analysis of the reaction products 46 Transformation of the reaction mixture 46 Analysis of transformants by DNA sequencing 47 References 47 3. Phosphorothioate-based site-directed mutagenesis for single-stranded vectors 49 J. Sayers and F. Eckstei n 1. Introduction 49 Development of the methodology 50 2. Mutagenesis procedure 50 Preparation of single-stranded template DNA 50 The mismatch oligonucleotide 5 4 Preparation of RF-IV DNA 5 5 Filtration through nitrocellulose 5 7 Nicking reaction 5 8 Gapping reaction 5 9 Preparation of the mutant homoduplex : the repolymerization step 62 Transfection 62 3. Monitoring the procedure and debugging 64 4. Modifications of the basic system 66 5. Scope and limitations of the procedure 67 References 68
4. Site-directed mutagenesis using gappedheteroduplex plasmid DNA 7 1 S. Inouye and M. Inouye 1. Introduction 7 1 2. The plasmid mutagenesis method 72 Preparation of plasmid DNA fragments I and II 72 Design and preparation of oligonucleotides 75 Denaturation and renaturation 76 Primer extension and transformation 77 Screening and confirmation of the mutations 78 3. Concluding remarks 8 1 References 82 5. Phosphorothioate-based double-strande d plasmid mutagenesis 83 D. B. Olsen and F. Eckstein 1. Introduction 83 2. Overview of the methodology 83 Preparation of the double-stranded DNA templates 84 Preparation of mutant heteroduplex 88 Creation of the mutant homoduplex 93 Transformation of the mutant DNA 96 3. Monitoring the procedure by agarose gel electrophoresis 97 4. Discussion 98 Acknowledgements 98 References 99 6. Linker scanning mutagenesis 10 1 B. Luckow and G. Schütz 1. Introduction 101 Definition of linker scanning mutagenesis 10 1 Different strategies for the construction of linker scannin g mutations 10 1 Alternative scanning mutagenesis procedures 102 2. Construction of linker scanning mutants 104 Apurination of plasmid DNA by formic acid 104 Generation of nicks and small gaps in apurinic DNA b y exonuclease III 105 Linearization of nicked or gapped plasmid DNA b y nuclease SI 106
Ligation of linkers 10 8 Generation of DNA pool I 11 1 Generation of DNA pool II 11 5 Removal of the kanamycin resistance fragment 11 7 Size fractionation of mutated inserts 11 9 Identification of putative linker scanning mutations on th e basis of the topoisomer pattern 123 3. General aspects of linker scanning mutagenesis 129 Selecting a suitable DNA fragment 129 Selecting a suitable vector 129 Selecting a suitable linker 130 Essential steps 130 Potential problems 13 0 Advantages 13 1 Time requirements 13 2 Acknowledgements 132 References 132 7. Random chemical mutagenesis and th e non-selective isolation of mutated DNA sequences in vitro 13 5 C. Walton, R. K. Booth, and P. G. Stockley 1. Introduction 13 5 2. Applications of random chemical mutagenesis 135 Random versus directed mutagenesis in vitro 135 3. Chemistry of random mutagenesis in vitro 136 Background to chemical mutagenesis in vitro 136 Nitrous acid mutagenesis 137 Hydroxylamine mutagenesis 137 Bisulphite mutagenesis 137 Hydrazine mutagenesis 137 Acid depurination of DNA 138 4. Practical considerations for random chemica l mutagenesis 13 8 Vector damage 13 8 Distribution of induced mutations 139 Considerations in the choice of target fragment(s) 139 Vectors for mutagenesis of single-stranded DNA 14 1 Mutagenic procedures 143 Regeneration of duplex DNA from treated single-strande d DNA 145 Amplification and enrichment of treated sequences 146 Nature of the mutations generated 148
5. Electrophoretic techniques allowing the enrichment o f mutants 14 9 Theory of denaturing gradient gel electrophoresis (DGGE) 150 Apparatus 15 1 Determination of optimum conditions for DGGE 15 3 Preparative denaturing gels 159 Analytical denaturing gradient gel electrophoresis 160 References 16 1 8. An enzymatic method for the complet e mutagenesis of genes 163 J. K. C. Knowles and P. Lehtovaara 1. Introduction 163 Other random mutagenesis methods 163 2. Principle of the method 164 Advantages of the method 16 5 3. Practical aspects of the method 16 6 Isolation of template DNA 16 6 Base-specific limited elongation 16 7 Misincorporation and elongation 17 0 Transformation 17 2 Mutant characterization 17 2 4. Conclusions 17 3 References 174 9. Spiked oligonucleotide mutagenesis 17 7 S. C. Blacklow and J. R. Knowles 1. Introduction 177 Overview and theory 177 Strategy for spiked oligonucleotide-directed mutagenesis 182 2. Materials 182 Buffers and solutions 182 Media 184 Bacterial strains 184 3. Procedures 185 Preparation of oligonucleotides for mutagenesis 185 Preparation of single-stranded template (for mutagenesis) 189 Oligonucleotide-directed mutagenesis 192 Preparation of competent cells and transformation 192 Determination of mutagenesis efficiency by sequencing 194 Amplification of mutant plasmid DNA 196
4. Summary 19 8 Acknowledgements 19 8 References 19 8 10. Cassette mutagenesis 19 9 J. H. Richards 1. Introduction 1 99 2. Requirement for restriction sites 200 3. Preparation of vector to receive a cassette 201 Restriction digest of plasmid DNA 2 02 4. Preparation of cassette for insertion 203 Discrete sequence cassette 204 Multiple sequence cassette 205 Preparation of double-stranded cassettes 208 5. Ligation 21 2 6. Transformation 21 3 References 21 4 11. Recombination and mutagenesis of DN A sequences using PCR 21 7 R. M. Horton and L. R. Pease 1. Introduction 21 7 The third generation of recombinant DNA technology 21 8 Synthetic applications of PCR 21 8 2. Overlap extension 218 Mechanism 218 Mutagenesis 222 Recombination 222 What happens to the non-productive strands? 225 3. Examples of overlap extension applications 226 Mutagenesis 226 Recombination 228 4. Planning a strategy 228 Is PCR the solution to your problem? 228 Oligonucleotide design 23 1 Establishing a cassette system 235 5. Procedures 236 Purifying the oligonucleotides 236 Determining the amount of primer to add 236