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Recombinant DNA can be cloned by using a vector to insert it into a suitable host cell. The vector often has a selectable marker or other reporter gene that gives the host cell a phenotype by which transgenic cells can be identified.
learning outcomes
You should be able to:
Compare and contrast different types of vectors and reporter genes.
Outline the steps involved in creating an organism or cell with recombinant DNA, including detection of the cells containing that DNA.
Compare the use of a GFP reporter gene with that of an antibiotic resistance gene in detecting cells harboring a foreign DNA sequence.
GFP reporter: all cells alive. Antibiotic resistance: most cells dead and only a selection alive. GFP reporter: need only a UV lamp to see the reporter gene product. Antibiotic resistance: need to alter growth medium and wait until nonselected cells are dead to identify cells with the vector.
Assume you are using a plasmid vector that contains genes encoding ampicillin resistance and the green fluorescent protein, with a restriction site in the latter gene only. Arrange a−e below to reflect the sequence of steps for inserting a piece of foreign DNA into this plasmid, introducing the recombinant plasmid into bacteria, and verifying that the plasmid and the foreign gene are both present in the bacteria.
Transform host cells.
Select colonies for antibiotic resistance.
Select colonies that do not glow under ultraviolet light.
Digest vector and foreign DNA with a restriction enzyme.
Ligate the digested plasmid together with the foreign DNA.
The sequence would be: d, e, a, b, c.
We have described how DNA can be cut with restriction enzymes, inserted into a vector, and introduced into host cells. We have also seen how host cells carrying recombinant DNA can be identified. Now let’s consider where the genes or DNA fragments used in these procedures come from.