A rapid and efficient method for site-directed mutagenesis using one- step overlap extension PCR
نویسندگان
چکیده
منابع مشابه
A 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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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...
متن کاملSite-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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Mutagenesis is an important tool to study gene regulation, model disease-causing mutations and for functional characterisation of proteins. Most of the current methods for mutagenesis involve multiple step procedures. One of the most accurate methods for genetically altering DNA is recombineering, which uses bacteria expressing viral recombination proteins. Recently, the use of in vitro seamles...
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BACKGROUND Mutagenesis plays an essential role in molecular biology and biochemistry. It has also been used in enzymology and protein science to generate proteins which are more tractable for biophysical techniques. The ability to quickly and specifically mutate a residue(s) in protein is important for mechanistic and functional studies. Although many site-directed mutagenesis methods have been...
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ژورنال
عنوان ژورنال: Nucleic Acids Research
سال: 1997
ISSN: 1362-4962
DOI: 10.1093/nar/25.11.2227