Recombinant DNA
Prof Peter Chantler
Course: Molecular Biology of the Cell
Year 1
Molecular Cloning
b. Insertion of the chosen DNA into a vector
The cloning of DNA requires the production of large quantities of the DNA of interest. This is accomplished by taking advantage of the fact that bacteria and bacteriophage replicate their DNA with high fidelity, many times, in a relatively short time-frame.
There are two sorts of cloning vector: viral vectors (known as bacteriophage, or simply, phage) and plasmid vectors.
Transformation
Typically, phage transformation is used when substantial replication of material is required, being up to 200-fold more efficient than plasmid vectors, the latter being used when the final step involves the preparation of pure DNA. Many contemporary vectors incorporate features from both phage and plasmids and are known as phagemid vectors.
The animation below encapsulates the processes involved in either plasmid or phage transformation. Play the animation to see a schematic of vector formation using a restriction enzyme. A recombinant vector DNA molecule containing foreign insert DNA is formed.
The principles involved in the use of both vector types are conceptually similar. The circular form is cleaved with a restriction nuclease giving a linear vector with cohesive ends. The insert DNA, cleaved by the same or compatible restriction enzyme, is incubated with the restricted vector, causing the two fragments to anneal. Covalent sealing utilizes DNA ligase, which to the 3' hydroxyl of the adjacent nucleotide on the other fragment.
Plasmid vectors
Plasmid vectors possess an origin of replication, ori, (a site where DNA replication initiates), a gene for antibiotic resistance, such as AmpR, which allows for selection of all cell clones carrying the plasmid, and a polylinker region which contains a variety of restriction sites from which to choose. The polylinker region is often located within the lacZ' gene, facilitating blue/white selection of bacterial colonies (the insert disrupts lacZ' transcription preventing a-complementation).
Phage vectors
Phage vectors (eg. lgt10) also possess a unique cloning site; in addition they possess complementary, cohesive (cos) ends - which may be regarded as giant sticky ends, operating on the same principle as the overlapping ends within overhanging restriction sites, but are much larger.
Course
1 Molecular Cloning
a Choice
b Insertion
c DNA Library
d Screening
e Exp Library
2 DNA Sequencing
3 PCR
4 VNTRs
5 RAPD
Contacts
Peter Chantler
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