A simple method for site-directed mutagenesis using the polymerase chain reaction (original) (raw)
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Careful titration of Vent polymerase activity allows efficient amplification of full-length plasmids (12 kb). The high processivity and fidelity of this enzyme made oligonucleotidedirected site-specific mutagenesis of plasmids a straight-forward process. Using only two primers, a mutagenic and a complementary, single-base mutants of recombinant plasmids were obtained consistently with >90% efficiency from a single round of PCR. This procedure also made site-specific deletion, insertion, and several bases mutagenesis facile and efficient. i Corresponding author. E-MAIL kgupta@rpslmc-edu; FAX (312) 226-6020. 404 ~ GENOME RESEARCH 5:404-407 ©1995 by Cold Spring Harbor Laboratory Press ISSN 1054-9803/95 $5.00 Cold Spring Harbor Laboratory Press on March 22, 2015 -Published by genome.cshlp.org Downloaded from PLASMID MUTAGENESIS enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487-491.
A simple and efficient method for site-directed mutagenesis with double-stranded plasmid DNA
Nucleic Acids Research, 1993
A general, simple and efficient method for preparing site-specific mutations in double-stranded plasmid DNA without the need for special plasmlds, bacterial strains or reagents Is described. Only one synthetic oligonucleotlde for each mutation is required, subclonlng Is unnecessary and a high efficiency of mutation (58-97%) was obtained. If two synthetic oligonucleotide primers are used, two separate mutations can be simultaneously created In a single reaction tube.
A novel PCR strategy for high-efficiency, automated site-directed mutagenesis
Nucleic Acids Research, 2005
We have developed a novel three-primer, one-step PCR-based method for site-directed mutagenesis. This method takes advantage of the fact that template plasmid DNA cannot be efficiently denatured at its reannealing temperature (T ra ), which is otherwise a troublesome problem in regular PCR. Two flanking primers and one mutagenic primer with different melting temperatures (T m ) are used together in a single PCR tube continuously without any intervention. A single-stranded mutagenic DNA (smDNA) is synthesized utilizing the high T m mutagenic primer at a high annealing temperature, which prevents the priming of the low T m primers (i.e. the two flanking primers). A megaprimer is then produced using this smDNA as the template at a denaturing temperature that prevents wild-type template DNA activity. The desired mutant DNA is then obtained by cycling again through these first two steps, resulting in a mutagenic efficiency of 100% in all tested cases. This highly automated method not only eliminates the necessity of any intermediate manipulation and accomplishes the mutagenesis process in a single round of PCR but, most notably, enables complete success of mutagenesis. This novel method is also both cost and time efficient and fully automated.
A fast and simple method for simultaneous mixed site-specific mutagenesis of a wide coding sequence
Biotechnology and Applied Biochemistry, 2008
Background site-specific mutagenesis at one or multiple sites has recently become an invaluable strategy in functional proteomic studies and genetic engineering. In the present paper we describe a novel PCR-based procedure for site-specific mutagenesis that permits, in a single-step, all three types of nucleotide sequence mutation (deletion, insertion and substitution). The entire procedure is carried out in one tube and takes about 3-4 h. The method utilizes two primers, one of which is phosphorylated at the 5 -terminus, that are designed to directly anneal back-to-back to the target sequence inserted in a plasmid. For the deletion type of mutagenesis (which has virtually no limit to its extent), primers anneal at the ends of the sequence to be deleted. For insertion and substitution types of mutagenesis the primers bear the mutagenic sequences in a tail. The entire circular plasmid, here tested for a maximum length of 7 kbp, is amplified by inverse PCR. The PCR product incorporates the desired mutagenesis and, after ligation, the plasmid is ready for cloning into bacteria. The method has been proved very efficient for deletions of up to 279 nucleotides, for introducing simultaneous deletions, insertions and substitutions, and for performing alanine scanning over a wide coding region. The procedure is suitable for applications in genetic engineering and for the construction of libraries.
A novel method for site-directed mutagenesis using PCR and uracil DNA glycosylase
Genome Research, 1992
A novel method for site-directed mutagenesis of DNA sequences based on the use of the PCR is described. The method uses two oligonucleotide primers that contain the desired sequence change and overlap at their 5' ends. In addition, the thymine residues in the overlap region have been substituted with deoxyuracil. Amplification of the template plasmid by PCR results in incorporation of the primers and the desired mutation into the PCR product. Excision of the deoxyuracil residues in the PCR products by uracil DNA glycosylase (UDG) destablizes base-pairing at the ends of DNA molecules and thus generates 3' protruding ends in the opposite strand. Due to overlapping nature of the primers, the resulting 3' protruding ends are complementary and can anneal rapidly after treatment with UDG. When the entire plasmid is amplified, a linear mutant PCR product is generated that circularizes after treatment with UDG. Circularized molecules can then be transformed into competent cells ...
Efficient strategy for introducing large and multiple changes in plasmid DNA OPEN
Scientific Reports, 2018
While the QuikChange site-directed mutagenesis method and its later modifications are extremely useful and simple, they suffer from several drawbacks. Here, we propose a new method, named LFEAP mutagenesis (Ligation of Fragment Ends After PCR) for creating various mutations in plasmid by leveraging three existing concepts: inverse PCR, single primer PCR, and sticky-end assembly. The first inverse PCR on the target plasmid yielded linearized DNA fragments with mutagenic ends, and a second single primer PCR resulted in complementary single-stranded DNA fragments with the addition of overhangs at the 5′ end of each strand. The resulting single strands were then annealed to produce double-stranded DNA with free 5′ single-stranded DNA tails. These products with compatible sticky ends were efficiently assembled into a circular, mutagenized plasmid. With this strategy, multiple simultaneous changes (up to 15) and mutations in large plasmids (up to 50 kb) were achieved with high efficiency and fidelity. LFEAP mutagenesis is a versatile method that offers significant advantages for introducing large and multiple changes in plasmid DNA. Polymerase chain reaction (PCR)-based site-directed mutagenesis is an invaluable technique for altering genes and hence the structure and activity of individual proteins in a systematic way, opening up opportunities for investigating the structure-function relationships of protein, enzyme specificity and selectivity, or protein engineering 1-3. In the past decade, a number of strategies and commercial kits have been developed for introducing muta-tional changes in plasmid DNA, such as base substitutions and base additions or deletions. Among them, Stratagene's QuikChange site-directed mutagenesis kit is extremely useful and simple, and probably one of the most favored 4. It requires a high-fidelity DNA polymerase that minimizes unwanted mutations, such as KOD hot start DNA polymerase, Pfu DNA polymerase, or Phusion ® high-fidelity DNA polymerase, to amplify the whole plasmid with complementary primer pairs, carrying the desired mutation in the form of mismatches to the original plasmid. The parental DNA template is eliminated by treating with DpnI, which destroys the methylated template DNA 5. The resulting nicked DNA is transformed into competent E. coli cells for nick repair. Despite its widespread use, the QuikChange system has limitations. The fact that the primers are completely complementary, and hence favor self-annealing limits the PCR product yield and gives rise to false positives 6. The complementary primer pairs favor "primer-dimer" formation by partial annealing of a primer with the second primer in the reaction, instead of primer annealing to the template with mismatches, which causes low PCR amplification efficiency, and may lead to the formation of tandem primer repeats in resulting PCR products and hence a reduction in fidelity 7,8. The complementary primer design results in the mutated plasmid containing staggered nicks, and thus the newly synthesized DNA cannot be used as a template for subsequent amplification 4. In addition, the originally developed QuikChange method requires the altered nucleotides to be introduced in the middle of both primers, limiting the introduction of multiple mutations 4 as well as large changes 9. To circumvent these limitations, many modified versions of the QuikChange site-directed mutagenesis method have been developed 4,10-12. These methods use partially overlapping primers to reduce the formation of primer dimers and hence improve PCR amplification efficiency. Despite high efficiency, these approaches require
Gene, 1992
A method for the oligodeoxyribonucleotide-directed mutagenesis of double-stranded DNA without the necessity for phenotypic selection is described. Plasmids denatured with alkali and purified by adsorption to and elution from nitrocellulose have single-stranded regions where primers can hybridize and serve as templates for a T7 DNA polymerase-catalyzed synthesis of complementary mutant DNA strands. When this procedure was carried out such that the original nonmutant strand contained uracil [method of Kunkel, Proc. Natl. Acad. Sci. USA 82(1985)488-4921, mutation frequencies of between 3O"/b and 40% were obtained. The technique has been used to generate mutant genes in plasmids of a wide variety of sizes. The largest plasmid manipulated and successfully mutagenized was 22 kb. The method is rapid and efficient and is not dependent upon either fl phage vectors or the presence of restriction sites in the vicinity of the sequence targeted for mutation.
Nucleic Acids Research, 1998
The QuikChange™ protocol is one of the simplest and fastest methods for site-directed mutagenesis, but introduces mutations at only one site at a time, and requires two HPLC-purified complementary oligonucleotides. Here, we describe that this method can be used with non-overlapping oligonucleotides. By doing this, two separate sites can be mutagenised simultaneously, or money can be saved by using a second 'standard' oligonucleotide. By a further modification, we have also used the QuikChange™ approach to exchange DNA sequences between closely related genes.