Why can you not use human DNA polymerase when you perform PCR?
Heat: PCR reactions are heat intensive processes, which require a polymerase that is able to withstand the heat. Taq polymerase is what is usually used in PCR reactions.
To understand why human DNA polymerase cannot be used in PCR (Polymerase Chain Reaction), it is essential to know the basic principles of PCR and the properties of DNA polymerases.
PCR is a technique used to amplify a specific DNA sequence by repeatedly and precisely copying it. This process involves three steps: denaturation, annealing, and extension, which occur in cycles.
During the denaturation step, the double-stranded DNA template is heated to separate the two DNA strands, resulting in single-stranded DNA.
In the annealing step, short DNA primers are added, which bind specifically to the desired DNA sequence on each strand.
Finally, in the extension step, DNA polymerase extends the primers, synthesizing new DNA strands in a temperature-dependent manner, using the original DNA strands as templates.
PCR requires a DNA polymerase enzyme that can withstand the high temperatures used in the denaturation step and efficiently synthesize DNA in the extension step. This is where human DNA polymerase falls short, as it is not suitable for PCR for several reasons:
1. Temperature sensitivity: Human DNA polymerases are optimally active around the body temperature of humans (37°C/98.6°F). PCR, on the other hand, utilizes high temperatures up to 95°C (203°F) during the denaturation step. Human DNA polymerase would denature or become non-functional at these elevated temperatures, preventing successful amplification.
2. Lack of processivity: Processivity refers to the capability of a DNA polymerase to continuously replicate a DNA template without dissociating from it. Human DNA polymerase has low processivity, making it inefficient for PCR, which requires rapid and continuous replication.
3. Enzyme purity and inhibitors: Human DNA polymerases obtained from natural sources often contain additional proteins or impurities that can interfere with PCR reactions. These impurities can inhibit the amplification process or lead to nonspecific DNA products.
To overcome these limitations, DNA polymerases derived from thermophilic bacteria or other extremophiles are used in PCR. These enzymes, such as Taq DNA polymerase, are highly heat-resistant, have high processivity, and possess optimal activity at high temperatures required for PCR.
Therefore, to perform PCR successfully, it is important to use DNA polymerases specifically engineered or selected for their suitability to withstand the temperature variations and fulfill the requirements of the PCR process.