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Medical biotechnology
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
Although these steps are invariable among cloning procedures, a number of alternative routes can be selected. These are summarized as a “cloning strategy.” Initially, the DNA of interest needs to be isolated to provide a DNA segment of suitable size. Subsequently, a ligation procedure is used, where the amplified fragment is inserted into a vector (piece of DNA). The vector (which is frequently circular) is linearized using restriction enzymes and incubated with the fragment of interest under appropriate conditions with an enzyme called DNA ligase. Following ligation, the vector with the insert of interest is transfected into cells. A number of alternative techniques are available, such as chemical sensitization of cells, electroporation, and biolistics. Finally, the transfected cells are cultured. As the aforementioned procedures are of particularly low efficiency, there is a need to identify the cells that have been successfully transfected with the vector construct containing the desired insertion sequence in the required orientation. Modern cloning vectors include selectable antibiotic resistance markers, which allow only cells in which the vector has been transfected to grow. Additionally, the cloning vectors may contain color selection markers that provide blue/white screening (-factor complementation) on X-gal medium.
Genomics and Bionanotechnology
Published in Anil Kumar Anal, Bionanotechnology, 2018
The general procedure of gene cloning involves in vitro assembly of recombinant DNA followed by insertion to host cell, which can direct the replication of recombinant DNA in coordination with its growth. Nonpathogenic bacterial strain of Escherichia coli is used as host cell, which can grow exponentially to generate virtually unlimited identical copies of the target DNA. Vectors are the DNA fragments with origin of plasmid replication and the DNA fragment with no origin of replication, which is being joined to vector is known as insert. Insertion into a cloning vector and the exchange of a vector DNA fragment for an insert DNA fragment are the standard DNA cloning approaches.
Glossary of scientific and technical terms in bioengineering and biological engineering
Published in Megh R. Goyal, Scientific and Technical Terms in Bioengineering and Biological Engineering, 2018
Cloning vector is a small, self-replicating DNA molecule – usually a plasmid or viral DNA chromosome – into which foreign DNA is inserted in the process of cloning genes or other DNA sequences of interest.
Cytoplasmic and periplasmic expression of recombinant shark VNAR antibody in Escherichia coli
Published in Preparative Biochemistry and Biotechnology, 2019
Herng C. Leow, Katja Fischer, Yee C. Leow, Katleen Braet, Qin Cheng, James McCarthy
pGEM-T (Promega, Madison, WI, USA) was used as the cloning vector. pET-28a (Novagen, Madison, WI, USA) was used to construct the expression vectors, also the backbone for vector pDSB-28Y construction. The restriction enzymes, SpeI, NcoI and XhoI, and T4 DNA ligase, were purchased from New England Biolabs Inc. (USA).