Site-Directed Mutagenesis: A Two-Step Method Using PCR and Dpn I
نویسندگان
چکیده
منابع مشابه
Site-directed mutagenesis: a two-step method using PCR and DpnI.
with impervious exteriors, such as shells or chitinous exoskeletons. Advantages to this method are high throughput, high success rate and enough template DNA for several different assays (FRE-PCR used only 5 μL of the 20-μL supernatant). Other extraction methods that were tried and discarded for low success rates included boiling in water or PCR buffer, grinding before boiling, grinding with co...
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A rapid method is described to efficiently perform site-directed mutagenesis based on overlap extension polymerase chain reaction (OE-PCR). Two template DNA molecules in different orientations relative to only one universal primer were amplified in parallel. By choosing a high dilution of mutagenic primers it was possible to run an overlap extension PCR in only one reaction without purification...
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Site-directed mutagenesis is a powerful tool for producing mutants to assess the importance of specific amino acid residues in a protein’s structure and/or function. We wanted to generate mutants of human ETS1 cDNA in the pET15b vector (Novagen, Madison, WI, USA) from which we had been producing wild-type protein for structural studies (11). Since we were subject to the constraints of this vect...
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The combined overlap extension PCR (COE-PCR) method developed in this work combines the strengths of the overlap extension PCR (OE-PCR) method with the speed and ease of the asymmetrical overlap extension (AOE-PCR) method. This combined method allows up to 6 base pairs to be mutated at a time and requires a total of 40-45 PCR cycles. A total of eight mutagenesis experiments were successfully ca...
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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 prime...
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ژورنال
عنوان ژورنال: BioTechniques
سال: 1997
ISSN: 0736-6205,1940-9818
DOI: 10.2144/97234bm05