Multiple site-directed mutagenesis via simple cloning by prolonged overlap extension
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...
متن کاملPatch cloning method for multiple site-directed and saturation mutagenesis
BACKGROUND Various DNA manipulation methods have been developed to prepare mutant genes for protein engineering. However, development of more efficient and convenient method is still demanded. Homologous DNA assembly methods, which do not depend on restriction enzymes, have been used as convenient tools for cloning and have been applied to site-directed mutagenesis recently. This study describe...
متن کاملSite-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...
متن کاملSite-Directed Mutagenesis in Human Granulocyte-colony Stimulating Factor, Cloning and Expression in Escherichia coli
Human granulocyte colony stimulating factor (hG-CSF) induces proliferation and differentiation of granulocyte progenitor cells. This glycoprotein is currently being used for treatment of neutropenia, in patients who have undergone bone marrow transplantation. So far, different researchers have tried to enhance hG-CSF biological activity and stability. In this study, Polymerase Chain Reaction (P...
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: BioTechniques
سال: 2020
ISSN: 0736-6205,1940-9818
DOI: 10.2144/btn-2019-0104