Cloning a gene involves isolating the DNA from an organism, inserting it into a cloning vector at a specific restriction enzyme site and transferring it to another organism. PCR can be used to amplify the DNA for insertion into the vector and in the process add restriction enzyme sites. This is done in cases where the restriction enzyme sites in the cloning vector:
- Are not found outside the gene of interest;
- Include DNA other than that of interest;
- Give a fragment too large to insert into the cloning vector.
The restriction sites are included close to the 5' ends of the primers used in the PCR.
In Figure 19 the gene of interest is amplified using PCR to introduce the restriction site BamHI. Following the PCR reaction the product is digested with BamHI and ligated to the cloning vector which has also been digested with BamHI.