The quality of the PCR products depends greatly upon the selection of primers. When constructing a primer, it is necessary to take into consideration the following items:
- Length: Is the primer long enough to efficiently anneal to the target site? Also, is it specific enough to bind only at the target site?
- Temperature: The composition of the primer determines its temperature for annealing (the greater the GC content, the higher the annealing temperature). Because we are using two primers (for the two sides of the DNA strand), it is important that the primer annealing temperatures are close to each other (that way, we donít have one primer annealing at 60 degrees, but another wonít efficiently anneal until 70).
- Self Complementarity: Does the primer complement itself? If so, you might have primers sticking to each other...definitely not what you want to accomplish.
- Secondary Structure: Will the primer create any secondary structures (hairpins and the like) with itself or other DNA?
It is important to note that the Taq polymerase we use for PCR has been genetically engineered so that it no longer has proofreading ability. Also, Taq polymerase leaves an extra A overhang as when it reaches the end of the DNA strand and falls off (so it generates 3í A overhangs).