Question

It's your first week working in the lab and unfortunately you program the PCR machine incorrectly so that your first three PCR experiments do not work as expected. However, from each of your mistakes you learn more about how PCR works! For each of the following scenarios, state what you think the results will be and briefly explain your reasoning.
a) The denaturation step was performed at 72 degree C.
b) The annealing step was performed at 96 degree C.
c)The annealing step was performed at 24 degree C.

Answer 1

Answer:

a) this temperature is too low to denature DNA

b) this temperature is too high and will denature DNA

c) this temperature is too low to anneal DNA  

 

Explanation:

The Polymerase Chain Reaction (PCR) is a technique widely used in molecular biology in order to replicate a single fragment of DNA millions of times. The PCR amplification consists of three steps repeated over several cycles: (1) denaturation, where double-stranded DNA strands are separated; (2) annealing, where specific primers (i.e., short DNA fragments) bind to flanking ends of the DNA sequence to be amplified; and (3) extension, where a thermostable DNA polymerase extends the 3' end of each primer sequence. The annealing temperature should be between 48 to 72°C because this temperature is associated with the melting temperature (Tm) of the primers. The extension step should be between 68 to 72°C since from this temperature the thermostable Taq polymerase can extend the primers in order to form new DNA strands. Therefore, the annealing temperature cannot exceed the extension temperature. Moreover, when the denaturation temperature should be around 95°C (it is important to note that when the denaturation step is too long the DNA can be degraded).