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Site Directed Mutagenesis
Product Listing
Application Overview
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:
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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
Troubleshooting tips for Q5 SiteDirected Mutagenesis Kit
Tips for commonly encountered challenges in
site-directed mutagenesis.
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. 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.
Figure 1: Site-specific mutagenesis proceeds in less than 2 hours.
The use of a master mix, a unique multi-enzyme KLD enzyme mix, and a fast polymerase ensures that, for most plasmids,
the mutagenesis reaction is complete in less than two hours.
Figure 2: Q5 Site-Directed Mutagenesis Kit Overview.
This kit is designed for rapid and efficient incorporation of insertions, deletions and substitutions into doublestranded
plasmid DNA. The first step is an exponential amplification using standard primers and a master mix fomulation 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 chemicallycompetent cells (provided).
Figure 3: Primer Design for the Q5 Site-Directed Mutagenesis Kit
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 SiteDirected 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, nonmutagenic 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.
Reference:
Kunkel, T.A. (1985) Proc Natl Acad Sci U.S.A. 82(2):488-492. PMID: 3881765
Learn More
Site-directed mutagenesis — experimental considerations
Improved methods for site-directed mutagenesis using NEBuilder HiFi DNA
Assembly Master Mix
Construction of an sgRNA-Cas9 expression vector via single-stranded DNA oligo
bridging of double-stranded DNA fragments
Featured Products
Q5® Site-Directed Mutagenesis Kit
NEBuilder® HiFi DNA Assembly Master Mix
See entire list »
Protocols for Site Directed Mutagenesis
Protocols
Publications
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 Protocol (E0554)
Q5® Site-Directed Mutagenesis Kit (Without Competent Cells) Quick Protocol (E0552)
Q5® Site-Directed Mutagenesis Kit Quick Protocol (E0554)
Time-Saver Protocol for Restriction Enzyme Digests
Legal Information
Publications related to Site Directed Mutagenesis:
1. 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 Noncanonical 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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This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New
England Biolabs, Inc (NEB).
While NEB develops and validates its products for various applications, the use of this product may require
the buyer to obtain additional third party intellectual property rights for certain applications.
For more information about commercial rights, please contact NEB's Global Business Development team at
[email protected].
This product is intended for research purposes only. This product is not intended to be used for therapeutic or
diagnostic purposes in humans or animals.
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