Question

1. Which physical parameter fluctuates continuously while the PCR machine is running?

2. Name the most unusual characteristic of the PCR enzyme Taq polymerase?

3. Why is PCR (performed on the genetic material of the pathogen) not the greatest test to show how infectious this pathogen is?

Answer 1

1. A typical PCR process takes place in an automated thermal cycler machine consisting of a series of 20–40 repeated cycles with consistent temperature changes. In each cycle, there are three steps involved& they are: denaturation step (94°C–98°C), annealing step (50°C–65°C), and elongation step (72°C). So, temperature is the physical parameter that fluctuates continuously while the PCR machine is running.

2. The most important characteristic of Taq polymerase enzyme is that it is a heat stable enzyme and by using the thermostable Taq enables running the PCR at high temperature (~60 °C and above), which facilitates high specificity of the primers and reduces the production of nonspecific products, such as primer dimer. Also, use of a thermostable polymerase eliminates the need to add new enzyme to each round of thermocycling. Thus, the use of Taq polymerase was the key idea that made PCR to make the DNA analysis.

3. However, since the universal PCR can detect almost all bacteria, including normal flora such as staphylococci on the skin. Discriminating the contaminants is difficult particularly when the specimens contain few bacteria. Therefore, the sensitivity and specificity of PCR assay for detection of pathogens may not be better than those of culture assay.