Q.In agarose gel electrophoresis, DNA molecules are separated on the basis of their:

In agarose gel electrophoresis, DNA molecules are loaded into wells at the negative end of the gel. Since DNA is negatively charged due to its phosphate backbone, all fragments move toward the positive electrode — so charge alone does not cause separation; it only provides the driving force. …

Agarose gel electrophoresis is a technique used to separate DNA fragments after they have been cut by restriction enzymes. To understand how separation happens, you first need to picture what DNA is like in solution. DNA is a negatively charged molecule — the phosphate groups in its backbone give it a uniform negative charge per unit length. This means that, regardless of the size of the fragment, the charge-to-mass ratio of DNA is essentially constant. A 100-base-pair fragment carries the same charge per unit length as a 1000-base-pair fragment.

Now, when an electric field is applied across the agarose gel, all DNA fragments begin to move toward the positive electrode (the anode) because of their negative charge. If charge alone were the deciding factor, every fragment would move at the same speed. But that is not what happens.

The key is the gel itself. Agarose is a polysaccharide that forms a porous, sponge-like matrix when it solidifies. Think of it as a sieve with pores of a certain size. As DNA fragments migrate through this matrix, smaller fragments can slip through the pores more easily, while larger fragments get tangled and move more slowly. So the separation is based on size — or more precisely, on the length of the DNA molecule. The gel acts as a molecular sieve, and the rate of migration is inversely proportional to the logarithm of the fragment size. …

Alternative Approach: A Physics-Style Elimination Using Charge Density

This question can be answered with a single physical fact about DNA's charge, applied

as an elimination filter across the four options -- a technique worth reusing whenever a

question asks "separated on the basis of ___" for a charged biomolecule.

Step 1: State the key physical fact.

Every nucleotide in a DNA strand contributes one phosphate group, so DNA's negative

charge is directly proportional to its length. This means the charge-to-length ratio (charge density) is the same for every DNA fragment, short or long.

Step 2: Test "charge only" (option a).

If separation depended on total charge alone, a longer fragment (more total negative

charge) would always move differently from a shorter one in a simple, predictable way

independent of the gel -- but the gel matrix itself is what actually slows larger

fragments down. Charge alone, without the sieving gel, does not explain size-based

banding. Eliminate.

Step 3: Test "charge-to-size ratio" (option c).

Since this ratio is constant for all linear DNA fragments (Step 1), it cannot be the

basis for separating fragments FROM each other -- there is no variation in this ratio …