NEETZoologyBiotechnology - Principles and Processes
A researcher programs a thermal cycler for a PCR reaction with the following temperature profile for 30 cycles: 94^ C for 1 minute 85^ C for 1 minute 72^ C for 2 minutes The PCR fails to amplify the gene of interest. What is the most likely reason for this failure?
Options
- AThe double-stranded DNA will fail to separate into single strands at 94^ C .
- BThe primers will fail to form stable hydrogen bonds with the template DNA at 85^ C .
- CThe Taq polymerase enzyme will denature and permanently lose its catalytic activity at 85^ C .
- DThe extension step will occur normally, but with a very high rate of mutation.
Correct answer
B. The primers will fail to form stable hydrogen bonds with the template DNA at 85^ C .
Step-by-step solution
The three steps of a PCR cycle are Denaturation, Annealing, and Extension. Step 1 ( 94^ C ) is the denaturation step, which successfully separates the double-stranded DNA. Step 2 ( 85^ C ) is intended to be the annealing step. However, the optimum temperature for primer annealing is typically between 40^ C and 60^ C . At a high temperature of 85^ C , the short oligonucleotide primers possess too much kinetic energy to form stable hydrogen bonds with the complementary regions of the template DNA. Without annealed pr