Error-prone PCR and effective generation of gene variant libraries for directed evolution.
Methods Mol Biol
; 1179: 3-22, 2014.
Article
em En
| MEDLINE
| ID: mdl-25055767
ABSTRACT
Any single-enzyme directed evolution strategy has two fundamental requirements the need to efficiently introduce variation into a gene of interest and the need to create an effective library from those variants. Generation of a maximally diverse gene library is particularly important when employing nontargeted mutagenesis strategies such as error-prone PCR (epPCR), which seek to explore very large areas of sequence space. Here we present comprehensive protocols and tips for using epPCR to generate gene variants that exhibit a relatively balanced spectrum of mutations and for capturing as much diversity as possible through effective cloning of those variants. The detailed library preparation methods that we describe are generally applicable to any directed evolution strategy that uses restriction enzymes to clone gene variants into an expression plasmid.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Biblioteca Gênica
/
Reação em Cadeia da Polimerase
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Evolução Molecular Direcionada
Idioma:
En
Ano de publicação:
2014
Tipo de documento:
Article