Multi-fragment site-directed mutagenic overlap extension polymerase chain reaction as a competitive alternative to the enzymatic assembly method
نویسندگان
چکیده
منابع مشابه
Site-directed mutagenesis by overlap extension using the polymerase chain reaction.
Overlap extension represents a new approach to genetic engineering. Complementary oligodeoxyribonucleotide (oligo) primers and the polymerase chain reaction are used to generate two DNA fragments having overlapping ends. These fragments are combined in a subsequent 'fusion' reaction in which the overlapping ends anneal, allowing the 3' overlap of each strand to serve as a primer for the 3' exte...
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The "megaprimer" method (1) based on polymerase chain reaction (PCR) is one of the simplest and most versatile procedures of site-specific in vitro mutagenesis available to date. The method utilizes three oligonucleotide primers and two rounds of PCR performed on a DNA template containing the cloned gene that is to be mutated. The rationale of the method is shown schematically in Fig. 1 where A...
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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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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 significan...
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There are two different co-expression systems including bicistronic; dual-vector or two-promoter to express two different genes simultaneously and also to study protein-protein interactions. Bicistronic system has disadvantages e.g. compared with two-promoter system. In this paper, a simple method based on spliced overlap extension by polymerase chain reaction (SOE-PCR) technique was demonstrat...
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ژورنال
عنوان ژورنال: Analytical Biochemistry
سال: 2014
ISSN: 0003-2697
DOI: 10.1016/j.ab.2013.09.021