Site-directed mutagenesis (SDM) is a method to create specific, targeted changes in double stranded plasmid DNA. There are many reasons to make specific DNA alterations (insertions, deletions and substitutions), including:
- To study changes in protein activity that occur as a result of the DNA manipulation.
- To select or screen for mutations (at the DNA, RNA or protein level) that have a desired property
- To introduce or remove restriction endonuclease sites or tags
SDM is an in vitro procedure that uses custom designed oligonucleotide primers to confer a desired mutation in a double-stranded DNA plasmid. Formerly, a method pioneered by Kunkel (Kunkel, 1985) that takes advantage of a strain deficient in dUTPase and uracil deglycosylase so that the recipient E. coli degrades the uracil-containing wild-type DNA was widely used. Currently, there are a number of commercially available kits that also require specific modification and/or unique E. coli strains (for example, the Phusion® Site-Directed Mutagenesis from Thermo and the GeneArt® system from Life). The most widely-used methods do not require any modifications or unique strains and incorporate mutations into the plasmid by inverse PCR with standard primers. For these methods, primers can be designed in either an overlapping (QuikChange®, Agilent) or a back-to-back orientation (Q5® Site-Directed Mutagenesis Kit) (Figure 1). Overlapping primer design results in a product that will re-circularize to form a doubly-nicked plasmid. Despite the presence of these nicks, this circular product can be directly transformed into E. coli, albeit at a lower efficiency than non-nicked plasmids. Back-to-back primer design methods not only have the advantage of transforming non-nicked plasmids, but also allow exponential amplification to generate significantly more of the desired product (Figure 2). In addition, because the primers do not overlap each other, deletions sizes are only limited by the plasmid and insertions are only limited by the constraints of modern primer synthesis. Currently, by splitting the insertion between the two primers, insertions up to 100 bp can routinely be created in one step using this method.
Before primers are designed, it is important to determine which mutagenesis workflow is to be used. Here we present a comparison of three commercially available kits (Figure 3) and a brief description of important features.
Before you plan your next SDM experiment, be sure to read through our list of important experimental considerations.
Kunkel, T.A. (1985) Proc Natl Acad Sci U.S.A. 82(2):488-492. PMID: 3881765
- 5 Minute Transformation Protocol using NEB 10-beta Competent E. coli (C3019)
- Double Digest Protocol with Standard Restriction Enzymes
- High Efficiency Transformation Protocol using NEB 10-beta Competent E. coli (High Efficiency) (C3019)
- KLD Enzyme Mix Reaction Protocol (M0554)
- Optimizing Restriction Endonuclease Reactions
- Protocol for Control Reaction (E0552)
- Protocol for Control Reaction (E0554)
- Protocol for Q5® Hot Start High-Fidelity 2X Master Mix
- Q5® Site-Directed Mutagenesis Kit (Without Competent Cells) Protocol (E0552)
- Q5® Site-Directed Mutagenesis Kit (Without Competent Cells) Quick Protocol (E0552)
- Q5® Site-Directed Mutagenesis Kit Protocol (E0554)
- Q5® Site-Directed Mutagenesis Kit Quick Protocol (E0554)
- Yafeng Li, Delu Song, Ying Song, Liangliang Zhao, Natalie Wolkow, John W Tobias, Wenchao Song, Joshua L Dunaief 2015. Iron-induced Local Complement Component 3 (C3) Up-regulation via Non-canonical Transforming Growth Factor (TGF)-β Signaling in the Retinal Pigment Epithelium. J Biol Chem. 290, PubMedID: 25802332, DOI: 10.1074/jbc.M115.645903
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