Figure 1.4. Gene cloning.

Figure 1.4

Gene cloning. In this example, a small amount of foreign DNA (a few nanograms) is digested with EcoRI. This foreign DNA can come from any source, the only requirement being that it contains the same restriction endonuclease recognition sites as the vector. Plasmid vector is also digested with EcoRI to create a linear DNA molecule. The “sticky” single-stranded ends of the foreign DNA can align and base-pair with the complementary “sticky ends” of the plasmid, after which DNA ligase covalently bonds foreign DNA to plasmid DNA. This recombinant DNA is introduced into E. coli by a process called transformation. Since the bacteria themselves are not resistant to ampicillin, growth in ampicillin will select only those bacteria that have taken up the plasmid DNA (which carries an ampicillin resistance gene). The plasmid contains a bacterial origin of replication so that as the bacterial culture grows, plasmids replicate resulting in several copies in each bacterium. When the culture has grown to sufficient size, plasmid DNA can be isolated biochemically, foreign DNA can be cut from the plasmid using EcoRI, and the resulting yield will often be milligrams of DNA, that is, greater than a 106-fold amplification.

From: Chapter 1, Molecular Biology

Cover of Holland-Frei Cancer Medicine
Holland-Frei Cancer Medicine. 5th edition.
Bast RC Jr, Kufe DW, Pollock RE, et al., editors.
Hamilton (ON): BC Decker; 2000.
© 2000, BC Decker Inc.

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