A simple and efficient method for site-directed mutagenesis with double-stranded plasmid DNA (original) (raw)
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
Gene, 1979
A synthetic oligodeoxyribonucleotide mismatched at a single nucleotide to a specific complementary site on wild-type circular ¢X174 DNA can be used to produce a defined point mutation after in vitro incorporation into closed circular duplex DNA by elongation with DNA polymerase and ligation followed by transfection of Escherichia coli . The present study is an investigation of the optimum conditions required for the oligodeoxyribonucleotide-primed reaction for production of transition and transversion mutations in ¢X174 DNA, using the large (Klenow) fragment of E. coli DNA polymerase I. Under optimum conditions up to 39% of the progeny of transfection are the desired mutant and significant mutation is observed using a heptadeoxyribonucleotide.
Rapid and efficient site-specific mutagenesis without phenotypic selection
Methods in enzymology, 1987
Several single-base substitution mutations have been introduced into the lacZa gene in cloning vector M13mp2, at 40-60% efficiency, in a rapid procedure requiring only transfection of the unfractionated products of standard in vitro mutagenesis reactions. Two simple additional treatments of the DNA, before transfection, produce a sitespecific mutation frequency approaching 100%. The approach is applicable to phenotypically silent mutations in addition to those that can be selected. The high efficiency, %10-fold greater than that observed using current methods without enrichment procedures, is obtained by using a DNA template containing several uracil residues in place of thymine. This template has normal coding potential for the in vitro reactions typical of site-directed mutagenesis protocols but is not biologically active upon transfection into a wild-type (i.e., ung') Escherichia coli host cell. Expression of the desired change, present in the newly synthesized non-uracil-containing covalently closed circular complementary strand, is thus strongly favored. The procedure has been applied to mutations introduced via both oligonucleotides and error-prone polymerization. In addition to its utility in changing DNA sequences, this approach can potentially be used to examine the biological consequences of specific lesions placed at defined positions within a gene.
Solid phase in vitro mutagenesis using plasmid DNA template
Nucleic Acids Research, 1990
Site-specific mutagenesis was accomplished using a solid support to generate single stranded vector and insert fragments which can be used to form gap-duplex plasmids through flanking, complementary double stranded regions. More than 80% mutants were obtained in both a single and a double primer approach. No special vectors or strains are needed and mismatch repair is avoided as the mutagenesis region is in a single stranded form when transformed into the Escherichia coli host cell. The fragments to be immobilized can be produced either by a polymerase chain reaction using general primers or by a sitespecific restriction followed by a fill-in reaction. This novel method is rapid, simple and flexible and well suited for both manual and semi-automated in vitro mutagenesis protocols.
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 ...
A simple vector modification to facilitate oligonucleotide-directed mutagenesis
Nucleic Acids Research, 1990
We describe a simple modification of commonly used single-stranded cloning vectors that permits the efficient recovery of mutant DNA molecules in oligonucleotide-directed mutagenesis experiments, even when the absolute efficiency of mutagenesis is very low. The modification consists of the insertion of a short synthetic DNA fragment into the vector's polylinker and permits the identification of mutant clones based on a standard chromogenic plate assay for bacterial colonies or phage plaques producing functional beta-galactosidase. Other useful properties of the original vector are retained in the modified version. In vitro mutagenesis reactions are carried out with two oligonucleotides, one to introduce the mutation of interest, and the second to correct a frameshift mutation introduced into the betagalactosidase gene of the modified vector. We have found that these two sequence changes are closely linked following transformation of an appropriate E. coli strain with the products of the in vitro mutagenesis reaction, and have thereby recovered desired mutations at a frequency of about 50% even when the overall mutagenesis efficiency is less than 1%. By alternately correcting and re-introducing the beta- galactosidase frameshift mutation, we have shown that multiple rounds of mutagenesis can be carried out on the same template with a high efficiency of mutant recovery in each step. Modifications similar or identical to those we describe here should be feasible for most commonly used single-stranded cloning vectors and should increase the usefulness of these vectors by providing an additional option for oligonucleotidedirected mutagenesis to be used in conjunction with or in lieu of other commonly used approaches.