COURSE

Introduction

Choice of DNA:
genomic DNA or cDNA


Insertion of the chosen DNA into a vector

Creation of a DNA library

Library screening

Creation of an expression library

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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 describes the process involved in phage transformation. Play the animation to see a schematic of vector formation using a restriction enzyme. A recombinant vector DNA molecule containing a chromosomal DNA insert is formed.

The principles involved in the use of both vector types is 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 joins the 5' phosphate of the terminal nucleotide of one fragment to the 3' hydroxyl of the adjacent nucleotide on the other fragment.



 

 


 

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Plasmid vectors

Plasmid vectors possess an origin of replication, ori, (a site where DNA replication initiates), a gene for antibiotic resistance, 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.

 



 

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.



 
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Copyright © 1999 RVC


Course published 10/11/99