Adding Restriction Sites using PCR
Method for Adding Restriction Sites to the Ends of DNA
It is often useful to amplify a region of DNA to contain new restriction sites on the ends to allow easier downstream cloning. When designing primers for this purpose it is important to add extra nucleotides to the 5' end of each primer to allow the restriction enzymes to cut the DNA efficiently. Most enzymes like a minimum of three nucleotides but some will require four.
It is also important to consider that the melting point (TM) of the primers will vary depending on the stage of the reaction. During the first two rounds of the PCR reaction the primers will only bind the target with a sub-section of the primer, however, once the new DNA (with restriction sites on the ends) has been produced after the first few rounds of the reaction, the primers will bind along their whole length (with a higher TM). Normally, this is not a problem and a single melting point can be used, but if the amplification is inefficient, perhaps start with a lower melting point (that of the section that binds the target DNA) for the first few rounds of the reaction.
Author: Dr Ryan Cawood