Question
Explain the principle of gel electrophoresis. Describe the procedure for separating DNA fragments. Why do smaller fragments move faster? Name two applications of this technique.
(NEET 2023, similar pattern)
Solution — Step by Step
Gel electrophoresis separates charged molecules based on their size and charge when placed in an electric field.
DNA is negatively charged (due to phosphate groups in the backbone). When an electric field is applied, DNA fragments migrate toward the positive electrode (anode).
The gel (agarose or polyacrylamide) acts as a molecular sieve — smaller fragments move faster through the pores because they face less resistance. Larger fragments get slowed down.
- Prepare the gel: Dissolve agarose in buffer, pour into a casting tray with a comb (creates wells)
- Load samples: Mix DNA fragments with a loading dye (for visibility + density) and pipette into the wells
- Run the gel: Connect to a power supply. DNA migrates from cathode (negative) to anode (positive). Run for 30-60 minutes
- Stain the gel: Remove the gel and stain with ethidium bromide (intercalates between DNA bases and fluoresces under UV light)
- Visualise: View under UV light. DNA bands appear as bright fluorescent lines
A DNA ladder (marker with known fragment sizes) is run alongside to estimate the sizes of unknown fragments.
The agarose gel has a network of pores. Larger DNA molecules have to squeeze through and navigate around the pores — they get tangled and slowed down. Smaller molecules slip through easily.
Since all DNA fragments have the same charge-to-mass ratio (each nucleotide adds one negative charge and roughly the same mass), size is the only variable determining migration speed. The result: fragments separate by size, with the smallest travelling farthest.
- DNA fingerprinting — comparing DNA profiles for forensics or paternity testing
- Checking restriction digestion — verifying that a restriction enzyme has cut DNA at the expected sites
- PCR product verification — confirming that PCR amplified the correct-sized fragment
- Southern blotting — first step before transferring DNA to a membrane for hybridisation
Why This Works
The technique works because it exploits two physical properties simultaneously: the uniform negative charge of DNA (which makes it move in an electric field) and the sieving effect of the gel matrix (which separates by size). Together, these create a simple, reliable method to sort DNA fragments — much like how a sieve sorts grains by size.
Ethidium bromide works as a stain because it slides between the stacked base pairs of DNA (intercalation) and fluoresces orange under UV light. Each band on the gel represents fragments of the same size.
Alternative Method — Agarose vs Polyacrylamide
For NEET: agarose gels are used for larger DNA fragments (100 bp to 25 kb) and are easier to prepare. Polyacrylamide gels (PAGE) are used for smaller fragments (5-500 bp) and give better resolution. DNA sequencing gels use polyacrylamide. For most NEET questions, agarose gel electrophoresis is the default — mention it unless specified otherwise.
Common Mistake
Students write that “DNA moves toward the cathode (negative electrode).” DNA is negatively charged, so it moves toward the anode (positive electrode). Think of it as: negatives are attracted to positives. The wells (where you load the DNA) are placed near the cathode end, and DNA migrates toward the anode. Getting the direction wrong shows a fundamental misunderstanding of the principle.