Subcloning can be extraordinarily easy or extremely difficult. The simplest case requires cleavage of the desired fragment from the DNA in which it is located with one or two restriction enzymes, cleavage of the plasmid that will serve as the ultimate recipient with the same enzymes, and ligation of the fragment to the plasmid. In a somewhat more complicated version of simple cloning, the investigator uses the polymerase chain reaction to amplify the DNA to be subcloned. In this case, restriction sites can be incorporated into the primers used for PCR, generating products with the desired restriction sites located near the ends of the newly synthesized DNA. After cleavage of those sites, the fragment is ready for insertion into an appropriately cut vector. The major disadvantage of the method is the need for sequencing the amplified material, because even the best of the thermostable polymerases makes mistakes that might render the DNA useless for the anticipated application.
