- the process by which multiple copies of a gene are produced. Gene cloning involves cutting genes out of a chromosome and inserting them into plasmids. Plasmids can then be placed into bacteria, which will divide, forming multiple copies of the gene. Bacteria will also be able to express the gene and form the gene product. • A good plasmid for gene cloning must have particular restriction sites and genes that make it easily detectable. • The plasmid and gene of interest must both be cut with the same restriction enzyme in order to create complimentary sticky ends. This joining together of sticky ends allows the gene to be easily inserted into the plasmids. • Plasmids with DNA insertions are placed into bacteria, which must then be screened for the presence of the gene of interest.

- In the diagram to the left, a bacterial plasmid that has a resistance gene (ampR) is cut with a particular restriction enzyme. At the cut site is the lacZ gene. - DNA, with the human insulin gene somewhere in it, is cut with the same restriction enzyme. The cut plasmids and the cut DNA are mixed together with the enzyme ligase, allowing sticky ends to connect and ligate. - The resulting plasmids are put into bacteria by adding a chemical such as calcium sulfate, which makes the membrane more soluble. - The bacteria are screened for the presence of the lasmid by being grown on a medium with ampicillin. The bacteria that have the plasmid will be resistant to ampicillin. - The bacteria are then screened for the presence of a gene inserted into the plasmid. If a gene has been inserted into the plasmid, then the lacZ gene (located at the cut site) will not be functional. If no gene has been inserted into the plasmid, then lacZ will be functional. If lacZ is functional in the plasmid, it will cause a color change in the medium if certain chemicals are used. Those bacteria that do not cause a color change are the ones that have a gene inserted into the plasmid. - Once the bacteria have been screened for the presence of the plasmid and for the presence of a gene insert in the plasmid, the bacteria are then screened for the specific gene that codes for insulin

In order for a particular restriction enzyme to be effective with a plasmid with a resistance gene, what must be true?

- There must be at least one recognition site on the plasmid. - There must not be a recognition site within the resistance gene on the plasmid.

In an experiment, an ampicillin-resistant plasmid vector was used to introduce a piece of foreign DNA into bacterial cells. How could you isolate only the bacterial cells containing the new DNA?

- Culture the cells in a medium containing ampicillin

7.1.3 Biotechnology: Gene Cloning Flashcards by Irina Soloshenko

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7.1.3 Biotechnology: Gene Cloning Flashcards

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