Site‐directed Mutagenesis Using PCR

P Jones, Zeneca Agrochemicals, Bracknell, UK

Published online: July 2003

DOI: 10.1038/npg.els.0003784

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
    Higuchi R, Krummel B and Saiki RK (1988) A general method of in vitro preparation and specific mutagenesis of DNA fragments – Study of protein and DNA interactions. Nucleic Acids Research 16: 7351–7376.
    book 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.

Related Sub-Topics:

Techniques and Tools in Clinical Genetics | Techniques and Tools in Molecular Biology | DNA Damage and Repair | Biochemical and Biophysical Techniques | Enzymes: Structure and Action Mechanism | Nucleic Acids: Structure, Function, Metabolism
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Article Title: Site‐directed Mutagenesis Using PCR
 
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]