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A student sets up a Polymerase Chain Reaction (PCR) mixture containing template DNA, primers, dNTPs, and Taq polymerase. However, the student incorrectly programs the thermal cycler to alternate only between 54^ C and 72^ C , completely omitting the 94^ C step. What will be the most immediate reason for the failure of DNA amplification in this setup?

Options

  1. ATaq polymerase will denature at 72^ C and lose its catalytic activity.
  2. BThe primers will bind to the double-stranded DNA but extension will not occur.
  3. CThe new DNA strands will be synthesized but will immediately degrade.
  4. DThe primers will fail to anneal because the template DNA remains double-stranded.

Correct answer

D. The primers will fail to anneal because the template DNA remains double-stranded.

Step-by-step solution

In a standard PCR cycle, the first step is denaturation, which requires heating the mixture to around 94^ C to break the hydrogen bonds between the two strands of the template DNA. If this high-temperature step is omitted and the cycle only alternates between 54^ C and 72^ C , the template DNA will remain double-stranded. Primers require single-stranded DNA to recognize and bind to their complementary sequences. Because the DNA remains double-stranded, the primers will fail to anneal during the 54^ C step. Without

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