Polymerase Chain Reaction

Polymerase Chain Reaction (PCR) is a technique by which you synthesize mass quantities of DNA in vitro.  For this reaction, the critical ingredients in the recipe are the DNA you wish to replicate, well designed primers (to anneal and provide the free 3’ hydroxyl needed for DNA synthesis), Taq polymerase, and abundant nucleotide triphosphates (NTP’s).  There are three steps, repeated several times to create a potentially enormous amount of DNA.

  • Denaturation: 94oC: By heating the mixture up to 94 degrees, we force the DNA to denature (unwind and become single stranded).
  • Annealing: 55oC: Cooling the mixture down to 55 degrees allows the primers to anneal to the DNA, meaning they stick to complementary sites on the DNA that we wish to replicate.
  • Extension: 72oC:  Raising the temperature to 72 degrees (which is the temperature optimum for Taq polymerase) starts the extension process, where Taq polymerase will work off the primers provided and generate a new DNA strand, making the DNA double stranded again.

With each cycle of these three steps, the amount of DNA doubles. It is also important to notice that for the first few iterations, the chromosomal DNA will be the predominant template used for replication; after a few iterations, the PCR products themselves will overwhelmingly be used for the replication (because they far outpopulate the original chromosomal DNA).