A single-stage polymerase-based protocol for the introduction of deletions and insertions without subcloning (original) (raw)
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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.
Silpakorn U Science & …, 2007
Generating either single or multiple point mutations and producing deletions and insertions into target DNA sequences are critical and widely used experimental procedures in molecular biological studies. This article presents a modified mutagenesis protocol based on an overlapping-extension PCR amplification method. The procedure benefits from the design of mutagenic primers to generate overlapping megaprimers without the need for intermediate purification to remove unused primers. Unused primers are diluted out during the successive amplification-extension reactions. A key success to this modified method is the use of two flanking primers after the overlapping extension reaction. The use of Pfu DNA polymerase increases, compared with Taq DNA polymerase, amplification accuracy. The proposed procedure represents a simple and efficient method that introduces many types of mutations into specific target DNA fragments and creates either hybrid DNA fragments or internal nucleotide deletions.
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.
Simplified one-tube “megaprimer” polymerase chain reaction mutagenesis
Analytical Biochemistry, 2005
Site-directed mutagenesis has become an essential tool in the investigation of gene structure and function as well as the products of gene expression, namely RNA and protein. Selected positions in a DNA sequence are changed, and the eVects of the mutations are studied in vitro and/or in vivo. Although many diVerent mutagenic strategies are available [1], the simplicity, cost eVectiveness, and accuracy of "megaprimer" mutagenesis have made it a widely used and popular approach [2-14]. A number of modiWcations have been introduced to the original protocol, but the core idea remains the same, based on two rounds of PCR using two Xanking primers and one internal primer that carries the desired mutation. Most strategies require intermediate gel puriWcation to remove leftover primers from the Wrst round of PCR ampliWcation. Gel puriWcation introduces its own problems given that it is time-consuming, costly, and often a source of product loss. In response to this limitation, a number of eVorts to skip the gel puriWcation step and to develop one-tube procedures have been made [7,9,10,14]. Unfortunately some shortfalls remain. One of the modi-Wed procedures [9] is based on the use of a restriction enzyme to remove competing template that would result in unmutagenized products and decreased eYciency. The requirement for speciWc restriction sites, however, can be complicated and can signiWcantly limit the Xexibility of this approach. In an alternate strategy [10], the melting temperature of the Xanking primers plays a critical role in which a low-melting temperature primer, used in the Wrst PCR ampliWcation step, is temperature inactivated
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 rapid and efficient method for multiple-site mutagenesis with a modified overlap extension PCR
Applied Microbiology and Biotechnology, 2005
A rapid and efficient method to perform site-directed mutagenesis based on an improved version of overlap extension by polymerase chain reaction (OE-PCR) is demonstrated in this paper. For this method, which we name modified (M)OE-PCR, there are five steps: (1) synthesis of individual DNA fragments of interest (with average 20-bp overlap between adjacent fragments) by PCR with high-fidelity pfu DNA polymerase, (2) double-mixing (every two adjacent fragments are mixed to implement OE-PCR without primers), (3) pre-extension (the teams above are mixed to obtain full-length reassembled DNA by OE-PCR without primers), (4) synthesis of the entire DNA of interest by PCR with outermost primers and template DNA from step 3, (5) post-extension (ten cycles of PCR at 72°C for annealing and extension are implemented). The method is rapid, simple and error-free. It provides an efficient choice, especially for multiple-site mutagenesis of DNAs; and it can theoretically be applied to the modification of any DNA fragment. Using the MOE-PCR method, we have successfully obtained a modified sam1 gene with eight rare codons optimized simultaneously.
Segment-specific mutagenesis: extensive mutagenesis of a lac promoter/operator element
Proceedings of the National Academy of Sciences, 1982
A method for highly efficient segment-specific mutagenesis is described. The method uses as target for sodium bisulfite mutagenesis the DNA single strands ofa DNA restriction fragment that had been separated by cloning into base-complementary regions of a pair of phage fd vectors. After repair synthesis in vitro, the mutagenized DNA fragment is recovered by cloning into a nonmutated plasmid vector and analyzed for sequence and by functional tests. By using this method, the nucleotide sequence of a 109-base pair restriction fragment containing the lac promoter/operator from Escherichia coli was extensively modified. More than 90% of the 235 isolates obtained showed a change in phenotype; all of 22 analyzed for their nucleotide sequence were found to carry multiple C -* T point mutations in up to 60% of the possible target positions. Nevertheless, few isolates showed major changes in promoter activity relative to the nonmutated promoter element, which indicates a high degree of flexibility in the promoter sequence.