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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

  1. AThe double-stranded DNA will fail to separate into single strands at 94^ C .
  2. BThe primers will fail to form stable hydrogen bonds with the template DNA at 85^ C .
  3. CThe Taq polymerase enzyme will denature and permanently lose its catalytic activity at 85^ C .
  4. 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

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