Direct Selection

To be able to select for a cloned gene it is necessary to plate the transformants onto an agar medium on which only the desired recombinants, and no others, can grow.

The only colonies that are obtained will therefore be ones that comprise cells containing the desired recombinant DNA molecule.

The simplest example of Direct Selection occurs when the desired gene specifies resistance to an antibiotic. As an example will will consider an experiment to clone the gene for kanamcycin resistance from plasmid R6-5.

This plasmid carries genes for resistances to four antibiotics: kanamycin, chloramphenicol, streptomycin and sulphonamide. The kanamycin resistance gene lies within one of the 13 EcoRI fragments (a).

To clone this gene the EcoRI fragments of R6-5 would be inserted into the EcoRI site of a vector such as pBR322. The ligated mix will comprise many copies of 13 different recombinant DNA molecules, one set of which carries the gene for kanamycin resistance.

Insertional inactivation cannot be used to select recombinants when the EcoRI site of pBR322 is used. This is becuase this site does not lie in either the ampicillin or the tetracycline resistance of this plasmid.

But this is immaterial for cloning the kanamycin resistance gene because in this case the coloned gene can be used as the selectable marker. Transformants are plated onto kanamycin agar, on which the only cells able to survice and produce colonies are those recombinants that contain the cloned kanamycin resistance gene(c).

Next: Extending the Scope of Direct Selection...