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Modern Genetic Analysis Chapter 10. Recombinant DNA Technology
Solved Problem

1. | In Chapter 3, we studied the structure of tRNA molecules. Suppose that you want to clone a fungal gene that encodes a certain tRNA. You have a sample of the purified tRNA and an E. coli plasmid that contains a single EcoRI cutting site in a tetR (tetracycline-resistance) gene, as well as a gene for resistance to ampicillin (ampR). How can you clone the gene of interest? See answer

Solution

You could use the tRNA itself, or a cloned cDNA copy of it, to probe for the DNA containing the gene. One method is to digest the genomic DNA with EcoRI and then mix it with the plasmid, which you also have cut with EcoRI. After transformation of an ampS tetS recipient, AmpR colonies are selected, indicating successful transformation. Of these AmpR colonies, select the colonies that are TetS. These TetS colonies will contain vectors with inserts, and a great number of them are needed to make the library. Test the library by using the tRNA as the probe. You can examine those colonies hybridizing with the probe further to find out how much of the tRNA sequence they contain.

Alternatively, you can subject EcoRI-digested DNA from organism X to gel electrophoresis and then identify the correct band by probing with the tRNA. This region of the gel can be cut out and used as a source of enriched DNA to clone into the plasmid cut with EcoRI.

See question

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