Isolation of Taq Polymerase and PCR
Before Taq polymerase was discovered, the polymerase chain reaction (PCR) was a long and systematic procedure. PCR can be used to amplify small pieces of DNA or to generate copies of DNA segments. In genetics, PCR is used worldwide to perform things such as cloning and sequencing to analyze genes for diseases and identification. PCR consists of three steps: denaturing step, annealing step, and the elongation step. In order to perform PCR, you need five main ingredients: polymerase, template strand of DNA, buffer, primers, and nucleotides.
Taq polymerase is a polymerase that is found in hot springs. It is considered “thermophilic” because it is able to withstand very high temperatures. These temperatures are so high that it surpasses the temperature at which proteins need to be denatured during PCR. Knowing this, Thomas D. Brock knew that Taq polymerase would be the perfect polymerase for PCR and was able to isolate and patent it. Taq polymerase is now used as the common polymerase during PCR because it is able to withstand the first denaturing step which is usually set to greater than 90°C.
Before Taq polymerase was isolated for PCR, polymerase had to be added after every step, making it very long and tedious to perform. This is especially true considering many cycles are run at a time to produce millions of copies of DNA. Using the five ingredients needed for PCR (including Taq polymerase), we can now go over the essential steps of this process.
Ingredients: Taq polymerase, nucleotides, DNA template strand, primers, and a buffer
- Denaturing step (>90°C): All of the ingredients are heated to a high temperature for more than 20 seconds to denature the DNA strand and separating them. This occurs by melting and disrupting the hydrogen bonds between the two strands. At the end of this step, there are only single stranded DNA strands in the tube.
- Annealing step (50-65°C): The temperature is lowered in this steps for about 20 seconds. This allows the primers to attach or “anneal” onto the single-stranded DNA template in which it closely matches. The Taq polymerase now binds onto the new primer-template strands and start to synthesize DNA.
- Elongation step (75-80°C): The temperature is slightly raised again depending on the type of polymerase being used. During this step, a new DNA complementary strand is synthesized to the template strand in a 5’ to 3’ manner. Based on polymerase knowledge, DNA polymerase polymerizes at least a thousand bases per minute at its optimum temperature. This allows for the amount of DNA template to double by the end of the first cycle of PCR.
For the DNA to generate exponentially, many cycles are performed to generate millions of copies of the first DNA template strand.