The Invention and Basic Principles of PCR

        Since the discovery of DNA in the 1950s, the demand for scientific developments regarding DNA has increased at a rapid rate. This demand was recognized by a scientist at Cetus Corporation in California, Kary Mullis, in 1984 (Bartlett et al pg. 3., Deshpande et al.). Mullis used concepts from techniques that had already existed and combined them to make a new technique called polymerase chain reaction (PCR). The technique of using short strands of DNA or oligonucleotides and a DNA polymerase to make new copies of DNA was already invented at the time but repeating this process over and over with other modifications creates the PCR method (Bartlett et al. pg. 3). By using PCR, an extremely small sample of DNA can be amplified to generate thousands to millions of copies of a particular sequence. The invention of PCR earned Mullis a Nobel Prize in chemistry (Bartlett et al. pg. 3, Deshpande et al.) and a Japan Prize Award (Deshpande et al.). 

       There are three basic steps in the PCR process: denaturation, annealing, and extension. PCR uses DNA polymerase to amplify the DNA sample (Bartlett et al. pg. 4, Deshpande et al.) The polymerase used in PCR today, Taq polymerase, requires nucleotide bases, primers which are attached to the bases, and a longer DNA strand to serve as a template. (Artika et al., Deshpande et al). The first step, denaturation, occurs at 90 to 97 degrees Celsius. When the sample strand is heated, it denatures and splits apart into two separate strands. Annealing occurs next at 50 to 60 degrees Celsius where the primers bind to the template strand to prepare for extension. Lastly, extension occurs at 72 degrees where the Taq polymerase creates a complementary strand using the ends of the annealed primers by adding nucleotides. This is repeated 25 to 30 times to produce a large quantity of the sample (Deshpande et al.). 

Figure 1: The PCR process with temperature (C) vs. time. D represents denaturation, A represents annealing, and E represents Extension. Provided by Deshpande et al.

       One serious drawback of Mullis' originally method was the use of DNA polymerase I. The original method of PCR used DNA polymerase found from the bacteria E. coli (Bartlett et al. pg. 4, Deshpande et al.). In Mullis' original method, DNA polymerase I would become deactivated after the denaturation stage due to the high heat. Because DNA polymerase I was inactive, more of it needed to be added in between each cycle (Bartlett et al. pg. 4). This made the process extremely time consuming and inefficient (Bartlett et al pg. 4, Deshpande et al.). Another scientist at Cetus, Henry Erlich, recognized this error and sought out to find a solution. Erlich's lab utilized a thermostable polymerase from a bacterium called Thermophilus aquaticus (Taq polymerase) (Barlett et al pg. 4). Taq polymerase is found in hot springs which reach temperatures of about 110 degrees Celsius, which is higher than the temperature required in the denaturation step (Deshpande et al.).