A simple and efficient method for site-directed mutagenesis with double-stranded plasmid DNA (original) (raw)
Related papers
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.
Site-directed mutagenesis of double-stranded DNA by the polymerase chain reaction
Gene, 1994
We have developed a facile procedure for rapid PCR-based site-directed mutagenesis of double-stranded DNA. Increasing the initial template concentration and decreasing the PCR cycles to 5-10 allows us to reduce the rate of undesired second-site mutations and dramatically increase the time savings. Following PCR, DpnI treatment is used to select against parental DNA molecules. The DpnI (target sequence 5'-Gm6ATC) is specific for methylated and hemimethylated DNA and is used to digest parental DNA and select for mutation-containing amplified DNA. DNA isolated from almost all common Escherichia coli strains is Dam methylated and therefore susceptible to DpnI digestion. Pfu DNA polymerase is used, prior to intramolecular ligation of the linear template, to remove any bases extended onto the 3' ends of the PCR product by Tuq DNA polymerase. The recircularized vector DNA incorporating the desired mutations is transformed into E. coli. This method can be used independently of any host strain and vector.
A simple method for site-directed mutagenesis using the polymerase chain reaction
Nucleic Acids Research, 1989
We have developed a general and simple method for directing specific sequence changes in a plasmid using primed amplification by the polymerase chain reaction (PCR). The method is based on the amplification of the entire plasmid using primers that include the desired changes. The method is rapid, simple in its execution, and requires only minute amounts of plasmid template DNA. It is significant that there are no special requirements for appropriately placed restriction sites in the sequence to be manipulated. In our system the yield of transformants was high and the fraction of them harboring plasmids with only the desired change was consistently about 80%. The generality of the method should make it useful for the direct alteration of most cloned genes. The only limitation may be the total length of the plasmid to be manipulated. During the study we found that the Taq DNA polymerase used for PCR adds on a single extra base (usually an A) at the end of a large fraction of the newly synthesized chains. These had to be removed by the Klenow fragment of DNA polymerase to insure restoration of the gene sequence.
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.
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.
Nucleic Acids Research, 1982
This paper presents a versatile and efficient procedure for the construction of oligodeoxyribonucleotide directed site-specific mutations in DNA fragments cloned into Ml3 derived vectors. As an example, production of a transition mutation in a clone of the yeast MATal gene is described. The oligonucleotide is hybridized to the template DNA and covalently closed double stranded molecules are generated by extension of the oligonucleotide primer with E. coli DNA polymerase (large fragment) and ligation with T4 DNA ligase. The resulting double stranded closed circular DNA (CC-DNA) is separated from unligated and incompletely extended molecules by alkaline sucrose gradient centrifugation. This purification is essential for production of mutants at high efficiency. Competent E. coli JM101 cells are transformed with the CC-DNA fraction and single stranded DNA is isolated from individual plaques. The recombinants are screened for mutant molecules by 1) restriction endonuclease screening for the loss of the Hinf I site in the target region, and 2) by dot blot hybridization using the mutagenic oligonucleotide as probe. Double stranded DNA is isolated from the mutant and the production of the desired mutation is verified by DNA sequencing. Efficiency of mutant production is in the range of 10-45% and no precautions to prevent mismatch repair are required.
Improved oligonudeotide site-directed mutagenesis using M13 vectors
Nucleic Acids Research, 1985
An improved method is described for the construction of mutations in M13 vectors using synthetic oligonucleotides. The DNA is first cloned into a novel M13 vector (based upon M13mp18 or M13mp19), which carries a genetic marker that can be selected against, such as an EcoK or EcoB site, or an amber mutation in an essential phage gene. In this "coupled priming" technique, one primer is used to construct the silent mutation of interest, and a second primer is used to eliminate the selectable marker on the minus strand. After primer extension and ligation, the heteroduplex DNA is transfected into a strain of E. coli which is repair deficient and selects against the plus strand marker. Over 50 mutants have been constructed with this approach, and the yields can be excellent (up to 70%). For the stepwise construction of mutations using separate rounds of mutagenesis, the EcoK and EcoB markers offer a particular advantage over the amber marker. They permit selection in each round, as it is possible to cycle between the two markers. However for construction of multiple mutations over a short region, long synthetic oligonucleotides with multiple mismatches to the template can offer an alternative strategy.
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