Questions: The final step in a Sanger DNA sequencing reaction is to run the DNA fragments on a gel. What purpose does this serve? It separates DNA fragments generated during the sequencing reaction based on one-nucleotide differences in their size. It separates DNA fragments based on their charge. It adds ddNTP to the end of each DNA fragment. It changes the length of the DNA fragments.

The answer is the first one: It separates DNA fragments generated during the sequencing reaction based on one-nucleotide differences in their size.

Explanation for each option:

1. It separates DNA fragments generated during the sequencing reaction based on one-nucleotide differences in their size.

   • This is correct. In Sanger sequencing, DNA fragments of varying lengths are generated, each terminating at a different nucleotide. Running these fragments on a gel allows them to be separated by size, with each band representing a fragment that ends at a specific nucleotide. This separation is crucial for determining the DNA sequence.

2. It separates DNA fragments based on their charge.

   • This is incorrect. While DNA fragments do have a negative charge due to their phosphate backbone, the primary purpose of running them on a gel in Sanger sequencing is to separate them by size, not charge. The charge is uniform across DNA fragments, so it does not provide the necessary resolution for sequencing.

3. It adds ddNTP to the end of each DNA fragment.

   • This is incorrect. The addition of ddNTPs (dideoxynucleotides) occurs during the sequencing reaction itself, not during the gel electrophoresis step. ddNTPs are used to terminate DNA synthesis at specific nucleotides, creating the fragments that are then separated on the gel.

4. It changes the length of the DNA fragments.

   • This is incorrect. The gel electrophoresis step does not change the length of the DNA fragments. It simply separates the pre-existing fragments based on their size.

In summary, the purpose of running DNA fragments on a gel in Sanger sequencing is to separate them based on size, allowing for the determination of the DNA sequence.