In Vitro Mutagenesis

TOTAL SYNTHESIS OF MUTANTS . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 424 Solid Phase Chemical Synthesis of Oligodeoxyribonucleotides .. . ..... 424 Assembly of Oligodeoxyribonucleotides into Double-Strand DNA 427 Mutation by Partial Gene Synthesis (Cassette Mutagenesis) 428 OLIGODEOXYRIBONUCLEOTIDE HETERODUPLEX MUTAGENESIS . . . . . . . . . . . . . . . 428 Oligonucleotide Mutagenesis Using Single-Strand Vectors 431 Oligonucleotide Mutagenesis Using Double-Strand Vectors ........ 433 Plasmids with a Filamentous Phage Origin of Replication 435 Selection and Enrichment of Mutants 435 Screening for Mutants 437 TARGETED RANDOM POINT MUTATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 438 Chemical Reagents that Change Base Pairing Specificity 439 Chemical Reagents that Eliminate Base Pairing. . . . ..... . 440 Enzymatic Incorporation of Nucleotide Analogs that Induce Mispairing ... 441 Enzymatic Misincorporation of Nucleotides.. 441 Random Point Mutations Using Long Synthetic Oligonucleotides with a Low Level of Misincorporuted Nucleotides ..... .... . ..... 443 Screening and Selection for Mutations Produced by Random Point Mutagenesis..... 444 DELETION AND INSERTION MUTAGENESIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444 Deletion Mutagenesis .... .... . . 444 Linker Insertion Mutagenesis . . . . 445 IN VITRO CONSTRUCTIONS THAT TARGET IN VIVO MUTAGENESIS . . .. . . . . . . . . 446 ALLELE REPLACEMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446 CHARACTERIZATION OF MUTANTS 447 In Situ Characterization of Mutants .. .... . .. . . . . .. . .. . . . . . . . . . . . . . . . . . . . . . . . ...... . . . . . . .... . . 447 Recovery of Genetic Functions Prior to Characterization 448 Reversion by Site-Specific Mutagenesis 448