Question

Harry was investigating the paternity of a child in his laboratory. While performing agarose gel electrophoresis, he observed that DNA samples remained static. He checked all other parameters of the apparatus, did some adjustments (A) in that experiment. Thankfully, some amount of the sample was still left using which he started another experiment (B). Interestingly, those adjustments worked. A and B are most likely to be

• A. A - slightly increasing the voltage, B - running another gel with increased pore size
• B. A - slightly decreasing the voltage, B - running another gel with increased pore size
• C. A - slightly increasing the voltage, B - running another gel with decreased pore size
• D. A - slightly decreasing the voltage, B - running another gel with decreased pore size

Answer 1

The correct option is A A - slightly increasing the voltage, B - running another gel with increased pore size

Agarose gel electrophoresis is a technique commonly utilised for the separation of a mixture of DNA (a negatively charged molecule) fragments of varying sizes on agarose gel (a natural biopolymer) matrix under the influence of electric field. DNA, being negatively charged, is forced to migrate towards the anode (positively charged terminal) in the electrophoresis unit.

The technique is regulated by certain factors such as voltage supply and the concentration of the gel.

• Voltage: Higher the voltage, the higher is the electric field strength between the two electrodes. Hence, higher is the force acting on the charged molecule (here DNA) to migrate. Hence, increasing voltage speeds up the movement of DNA molecules, provided all other experimental parameters are kept constant.

Since the DNA fragments were not moving, Harry might have slightly increased the voltage in the experimental setting keeping the rest of the experimental parameters unaltered.

• Concentration of agarose in the gel: The concentration of agarose determines the pore size of the gel. The pores present in the agarose gel create a sieving effect. This helps in the separation of DNA molecules based on their size. For a specific DNA molecule, a gel with higher agarose concentration has smaller pore size and retards its movement compared to a gel with lower agarose concentration.

Since the DNA was not migrating significantly in Harry’s initial experiment, hence, it might have been due to the smaller pore size of the gel. The agarose concentration could have been too much resulting in excessive cross linking. This might have given rise to pores too small to allow the DNA molecules to pass through them. Hence in his second experiment, Harry might have casted a gel with relatively lesser concentration of agarose to have comparatively larger sized pores, keeping the rest of the experimental parameters constant.

Therefore, Harry must have increased the voltage in his initial experiment and ran another gel with increased pore size in his next experiment.