Q.With reference to the set-ups (A, B and C) given below, of the electrophoretic separation of a mixture of DNA fragments of varied lengths, answer the questions that follow : [Figure: Set-up A — DNA fragments in well near Cathode (top), Anode at bottom, current applied downward; Set-up B — well near Anode (top), Cathode at bottom, current applied downward; Set-up C — well at top with two separated bands labelled I (upper) and II (lower)]

Concept: DNA Electrophoresis (separation based on charge and size).

(a) DNA molecules are negatively charged due to the phosphate groups in their backbone. In an electric field, these negatively charged fragments migrate towards the positive electrode (anode). Set-up A places the DNA fragments in wells near the cathode (negative electrode) and the anode at the bottom, allowing the DNA to move downwards into the gel and separate. Set-up B places the wells near the anode, which would not facilitate migration into the gel for separation. …

DNA gel electrophoresis is a fundamental technique used to separate DNA fragments based on their size. The entire process relies on two key principles: the inherent electrical charge of DNA and the sieving effect of the gel matrix. Understanding these principles is crucial to determining the correct setup and interpreting the results.

First, DNA molecules possess a net negative charge due to the phosphate groups in their sugar-phosphate backbone. When placed in an electric field, these negatively charged DNA molecules will migrate towards the positive electrode, which is called the anode.

Second, the gel itself (typically agarose or polyacrylamide) acts like a molecular sieve. It contains a network of pores. Smaller DNA fragments can navigate through these pores more easily and quickly than larger fragments. Consequently, smaller fragments travel further down the gel in a given amount of time, while larger fragments are retarded and remain closer to the starting wells.

Let's apply these concepts to the given set-ups.

Part (a): Identifying the Correct Set-up for DNA Separation

1. Determine the direction of DNA migration:

   DNA is negatively charged. In an electric field, negative charges are attracted to the positive pole (anode) and repelled by the negative pole (cathode). Therefore, for DNA fragments to move into and through the gel for separation, they must be loaded near the cathode and migrate towards the anode.

2. Analyze Set-up A:

   In Set-up A, the wells (where DNA is loaded) are near the cathode (negative electrode) at the top, and the anode (positive electrode) is at the bottom. When current is applied, the negatively charged DNA fragments will be pulled downwards from the wells towards the positive anode, entering the gel and beginning their separation. This is the correct orientation for DNA electrophoresis.

3. Analyze Set-up B:

   In Set-up B, the wells are near the anode (positive electrode) at the top, and the cathode (negative electrode) is at the bottom. If DNA were loaded here, the negatively charged fragments would be repelled by the anode and attracted to the cathode. This would cause them to migrate upwards, away from the main body of the gel, or prevent them from entering the gel effectively for separation in the intended downward direction.

   Watch out

   A common mistake is to forget the charge of DNA or confuse the anode and cathode. Always remember: DNA is negative, so it moves towards the positive anode.

Therefore, Set-up A is the one where DNA fragments would be separated.

Part (b): Identifying Longer DNA Fragments in Set-up C

1. Recall the principle of separation by size: …