Question

What would the expected effect be on a PCR reaction if the primers used were slightly shorter and more variable than the intended oligonucleotide sequences?

The reaction would yield a mixture of non-specific products.
The PCR reaction would not commence.
The PCR reaction would end after one cycle.
The reaction would generate a single short PCR product.

Answer 1

Shorter and more variable primers in a PCR reaction will likely lead to non-specific binding and the production of a mixture of multiple non-specific products due to decreased annealing specificity.

Step-by-step explanation:

If the primers used in a PCR reaction are slightly shorter and more variable than the intended oligonucleotide sequences, the expected outcome is that the reaction will yield a mixture of non-specific products. Primers need to be complementary to the target DNA sequence to ensure specificity. When primers are not perfectly matched, they may bind to several sites leading to amplification of unintended sequences. Moreover, shorter primers are more prone to bind non-specifically as there are fewer base pairs to provide stable binding, increasing the likelihood of non-specific annealing.

During PCR amplification, specificity is crucial for producing the correct product. In the initial cycles of PCR, you may not get the desired DNA segment, and it is only after the second cycle that you typically amplify the specific DNA fragment you want. With more variable primers, the annealing process may not be as specific, and the resulting blend of amplified DNA may contain numerous unwanted sequences.