Q.Observe the given picture carefully. A mixture of DNA with fragments ranging from 100 base pairs to 1800 base pairs were separated by electrophoresis on agarose gel with the following arrangement :

Here is the explanation for the given electrophoresis setup:

1. When stained with ethidium bromide, the DNA fragments will appear as bright orange bands under ultraviolet (UV) light. Ethidium bromide is a fluorescent dye that intercalates, or inserts itself, between the base pairs of DNA. When this stained DNA is exposed to UV radiation, the ethidium bromide molecules fluoresce, making the DNA visible. Since the DNA fragments range from 100 base pairs to 1800 base pairs, multiple distinct bands will be observed, each representing a group of DNA fragments of a particular size.
2. To obtain a band with 100 base pairs at position 'X' in the modified setup:

• The gel was inverted, placing the wells at the bottom of the gel.
• The polarity of the electrodes was reversed: the negative electrode (cathode) was placed at the bottom, near the wells, and the positive electrode (anode) was placed at the top, where 'X' is located. …

Gel electrophoresis is a fundamental technique used to separate DNA fragments based on their size. DNA, being negatively charged due to its phosphate backbone, migrates towards the positive electrode (anode) when an electric field is applied across an agarose gel. The gel acts as a molecular sieve, impeding the movement of larger fragments more than smaller ones.

(a) Result of Staining with Ethidium Bromide

When the agarose gel, after electrophoresis, is stained with ethidium bromide, all the DNA fragments present in the gel will become visible as distinct bands under ultraviolet (UV) light.

• Ethidium Bromide's Action: Ethidium bromide is a fluorescent dye that has the ability to intercalate, or wedge itself, between the stacked base pairs of the DNA double helix. This binding is non-specific, meaning it binds to all DNA fragments regardless of their sequence or size.
• Fluorescence under UV Light: Once bound to DNA, ethidium bromide absorbs UV radiation and re-emits it as visible orange-red light. This property makes the DNA-ethidium bromide complex fluorescent.
• Visibility of Bands: Therefore, when the stained gel is placed on a UV transilluminator, all the separated DNA fragments, ranging from 100 base pairs to 1800 base pairs, will fluoresce and appear as bright orange-red bands. The intensity of a band is generally proportional to the amount of DNA present in that band.

(b) Changes Made and Reason for Band at 'X'

The setup was modified to obtain a band with 100 base pairs at 'X'. This implies that 'X' represents the furthest point of migration on the gel, closest to the positive electrode.

Why the band appeared at 'X':

The fundamental principle of agarose gel electrophoresis dictates that DNA fragments migrate through the gel matrix at different rates based on their size.

• Negative Charge of DNA: All DNA fragments carry a net negative charge, causing them to move towards the positive electrode.
• Size-Dependent Migration: Smaller DNA fragments encounter less resistance from the pores of the agarose gel matrix compared to larger fragments. Consequently, smaller fragments travel faster and migrate further down the gel towards the positive electrode.
• Smallest Fragment: In the given mixture, the 100 base pair fragments are the smallest. Therefore, they will exhibit the highest mobility and migrate the furthest distance on the gel, appearing at the position marked 'X'.

Changes made to the previous design to get a band at 'X': …