Questions: Although they share many approaches in common, there are several fundamental differences between the polymerase chain reaction (PCR) and Sanger (dideoxy chain termination) sequencing. What are these important differences?

The answer is a combination of the second, third, and sixth statements:

1. PCR generates a large number of "short" fragments and a small number of "intermediate" fragments from the template, whereas Sanger sequencing generates a mixed population of all fragment lengths possible from the template.
2. PCR uses a primer pair (i.e., forward and reverse) whereas Sanger sequencing uses just a single primer (e.g., forward).
3. Both use deoxynucleotides (dNTPs), but Sanger sequencing also requires modified dideoxynucleotides (ddNTPs).

Explanation for each option:

1. Unlike synthesis in PCR, Sanger sequencing proceeds in the $3^{\prime}$ to $5^{\prime}$ direction:

   • Incorrect. Both PCR and Sanger sequencing proceed in the $5^{\prime}$ to $3^{\prime}$ direction.

2. PCR generates a large number of "short" fragments and a small number of "intermediate" fragments from the template, whereas Sanger sequencing generates a mixed population of all fragment lengths possible from the template:

   • Correct. PCR amplifies specific regions of DNA, resulting in many copies of short fragments. Sanger sequencing, on the other hand, produces a range of fragment lengths due to the random incorporation of ddNTPs.

3. PCR uses a primer pair (i.e., forward and reverse) whereas Sanger sequencing uses just a single primer (e.g., forward):

   • Correct. PCR requires two primers to amplify both strands of the DNA, while Sanger sequencing uses a single primer to initiate the synthesis of the complementary strand.

4. Unlike PCR, Sanger sequencing does not require primers:

   • Incorrect. Sanger sequencing does require a primer to initiate DNA synthesis.

5. PCR uses DNA polymerase, but Sanger sequencing uses RNA polymerase:

   • Incorrect. Both PCR and Sanger sequencing use DNA polymerase.

6. Both use deoxynucleotides (dNTPs), but Sanger sequencing also requires modified dideoxynucleotides (ddNTPs):

   • Correct. PCR uses only dNTPs for DNA synthesis, while Sanger sequencing uses both dNTPs and ddNTPs to terminate DNA synthesis at specific points.

In summary, the important differences between PCR and Sanger sequencing are related to the types of fragments generated, the number of primers used, and the use of ddNTPs in Sanger sequencing.