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Home › Applications › Cloning & Synthetic Biology › Site Directed Mutagenesis 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: Videos 1 of 1 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 Legal and Disclaimers 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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