Mutagenesis

Importance of mutagenesis

Generation of mutants

• Chemical or physical agents

• Disadvantages – Lack of specificity

Site directed mutagenesis

• Basic tool of gene manipulation

• Highly specific

Primer extension

(Single Primer method):

Limitations of the method

• Rxn mixture is contaminated with non-mutant ssDNA template and some partially dsDNA
molecules.

• Replication provides both mutant and non-mutant progeny.

• Recovery of mutant progeny is low due to methyl-directed mismatch repair system in E.coli.

• Requirement of ssDNA as a template.

Primer extension

(Single Primer method)

Gene EditorTM system
SITE DIRECTED MUTAGENESIS BY PCR :