Site‐directed Mutagenesis Using PCR

Abstract

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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References

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Jones P (1995) Gel Electrophoresis: Nucleic Acids – Essential Techniques. Wiley: New York.

Ling M and Robinson BH (1995) A one‐step method polymerase chain‐reaction site‐directed mutagenesis method for large gene‐cassettes with high‐efficiency, yield and fidelity. Analytical Biochemistry 230: 167–172.

Marini F, Naeem A and Lapeyre J‐N (1993) An efficient 1‐tube PCR method for internal site‐directed mutagenesis of large amplified molecules. Nucleic Acids Research 21: 2277–2278.

Sarkar G and Sommer SS (1992) Double‐stranded DNA segments can efficiently prime the amplification of human genomic DNA. Nucleic Acids Research 20: 4937–4938.

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