Site‐directed Mutagenesis Using PCR


PCR‐based site‐directed mutagenesis is used to study gene expression, protein structure–function relationships, and vector modification. This technique uses a three‐stage PCR with optimized parameters for each stage and the use of a ddNTP‐blocked fragment to produce a final mutant product.

Keywords: gene expression; structure–function relationships; vector modification; three‐stage PCR

Figure 1.

Site‐directed mutagenesis using PCR. (a) Stage 1: megaprimer synthesis. (b) Stage 2: synthesis of a full‐length mutant strand. (c) Stage 3: synthesis of mutant duplexes.

Figure 2.

Restriction of DNA template to produce a blocked template for megaprimer synthesis.



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Jones, P(Jul 2003) Site‐directed Mutagenesis Using PCR. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1038/npg.els.0003784]