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Site Directed Mutagenesis

Site Directed Mutagenesis

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

Method Overview:

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.

Multi-site Mutagenesis

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.

Image of a workflow — This kit is designed for rapid and efficient incorporation of insertions, deletions and substitutions into double stranded plasmid DNA. The first step is an exponential amplification using standard primers and a master mix formulation of Q5 Hot Start High-Fidelity DNA Polymerase. The second step involves incubation with a unique enzyme mix containing a kinase, a ligase and DpnI. Together, these enzymes allow for rapid circularization of the PCR product and removal of the template DNA. The last step is a high-efficiency transformation into chemically competent cells (kit available with and without competent cells).

Image of primer design — Substitutions, deletions and insertions are incorporated into plasmid DNA through the use of specifically designed forward (black) and reverse (red) primers. Unlike kits that rely on linear amplification, primers designed for the Q5 Site-Directed Mutagenesis Kit should not overlap to ensure that the benefits of exponential amplification are realized. A) Substitutions are created by incorporating the desired nucleotide change(s) (denoted by *) in the center of the forward primer, including at least 10 complementary nucleotides on the 3´side of the mutation(s). The reverse primer is designed so that the 5´ ends of the two primers anneal back-to- back. B) Deletions are engineered by designing standard, non-mutagenic forward and reverse primers that flank the region to be deleted. C) Insertions less than or equal to 6 nucleotides are incorporated into the 5´ end of the forward primer while the reverse primer anneals back-to-back with the 5´ end of the complementary region of the forward primer. D) Larger insertions can be created by incorporating half of the desired insertion into the 5´ ends of both primers. The maximum size of the insertion is largely dictated by oligonucleotide synthesis limitations.

Workflow for multi site-directed mutagenesis using NEBuilder HiFi — *Using NEBuilder HiFi DNA Assembly (NEB #E2621), perform multi-site mutagenesis or combinatorial mutagenesis for diverse multi-site mutant library creation and screening.*

Reference:

Kunkel, T.A. (1985) Proc Natl Acad Sci U.S.A. 82(2):488-492. PMID: 3881765

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Protocols for Site Directed Mutagenesis

Application Notes for Site Directed Mutagenesis

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Molecular Cloning Technical Guide

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Troubleshooting Guide for Cloning

Publications related to Site Directed Mutagenesis

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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