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. The most widely-used methods do not require any specific modifications or unique E. coli strains and incorporate mutations into the plasmid by inverse PCR with standard primers. For these methods, primers can be designed in either an overlapping or a back-to-back orientation (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 (as used in NEB’s Q5® Site-Directed Mutagenesis Kit (NEB #E0554)) not only have the advantage of transforming non-nicked plasmids, but also allow exponential amplification, generating significantly more of the desired product (Figure 1). 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 four different primer designs (Figure 2) and the types of mutations they enable.
Before you plan your next site-directed mutagenesis experiment, be sure to read through our list of important experimental considerations.
Efficient and easy multi-site mutagenesis can be accomplished by using assembly methods such as NEBuilder® HiFi DNA Assembly (Figure 3). Learn more with our application note.
Kunkel, T.A. (1985) Proc Natl Acad Sci U.S.A. 82(2):488-492. PMID: 3881765
- Protocol for Control Reaction (E0554)
- Protocol for Q5® Site-Directed Mutagenesis Kit (E0554)
- Quick Protocol for Q5® Site-Directed Mutagenesis Kit (E0554)
- Protocol for Control Reaction (E0552)
- Protocol for Q5® Site-Directed Mutagenesis Kit (Without Competent Cells) (E0552)
- Quick Protocol for Q5® Site-Directed Mutagenesis Kit (Without Competent Cells) (E0552)
- KLD Enzyme Mix Reaction Protocol (M0554)
- 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, 11918-34. PubMedID: 25802332, DOI: 10.1074/jbc.M115.645903
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Learn how to create substitutions, deletions or insertions in 3 easy steps with the Q5 Site-Directed Mutagenesis Kit.
Tips for commonly encountered challenges in site-directed mutagenesis.