Improved site-directed mutagenesis method using PCR
نویسندگان
چکیده
منابع مشابه
Combined Overlap Extension PCR Method for Improved Site Directed Mutagenesis
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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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...
متن کاملSite-directed mutagenesis using PCR-mediated introduction of silent mutations.
The alteration of a specific sequence of DNA using site-directed mutagenesis (SDM) provides an effective tool to probe the structure and function of gene products. With the advent of polymerase chain reaction (PCR), several new techniques were developed that facilitate SDM. An oligonucleotide containing mismatches at the desired site of mutation is used to generate a population of mutant fragme...
متن کاملA novel method for site-directed mutagenesis using PCR and uracil DNA glycosylase.
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...
متن کاملA rapid and efficient method for site-directed mutagenesis using one-step overlap extension PCR.
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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ژورنال
عنوان ژورنال: Nucleic Acids Research
سال: 1991
ISSN: 0305-1048,1362-4962
DOI: 10.1093/nar/19.16.4558