10 min readPathway sheet1-mark MCQ, a 2 or 3-mark question on gel electrophoresis, and case-based questions on band patterns; also a NEET favourite

Cutting DNA and gel electrophoresis

Purified DNA is cut with a restriction enzyme. Agarose gel electrophoresis separates the fragments by size; smaller fragments move farther towards the anode.

MatrixAgarose, a natural polymer extracted from seaweeds

DirectionDNA is negatively charged, so it moves towards the anode (positive electrode)

Seeing DNAStain with ethidium bromide and expose to UV light: bright orange bands

ElutionCutting a band out of the gel and extracting the DNA from the gel piece

Cutting the source DNA and the vector

1
Incubate the purified DNA with a restriction enzyme under the conditions best for that enzyme.
2
Check how far the digestion has gone by agarose gel electrophoresis.
3
Cut the vector DNA with the same restriction enzyme.
4
Mix the cut-out gene of interest with the cut vector and add DNA ligase.
DNA ligase
5★
The matching sticky ends pair and ligase seals them: recombinant DNA is formed.

★ key step

Gel electrophoresis: how it works

1
Load the DNA fragments into wells at one end of an agarose gel, the end near the cathode (negative electrode).
2
Switch on the current. DNA is negatively charged, so it moves towards the anode.
3
The agarose acts as a sieve. Smaller fragments move faster and farther; larger ones stay nearer the wells.
4★
Stain the gel with ethidium bromide and expose it to UV light. DNA shows as bright orange bands. Pure DNA cannot be seen in visible light without staining.
5
Cut out the wanted band and extract the DNA from the gel piece: elution.
6
Use the purified fragment to build recombinant DNA with a cloning vector.

★ key step

Undigested vs digested DNA on a gel

Undigested DNAOne large molecule, so it gives a single band that stays near the well.

Digested DNACut into pieces of different sizes, so it gives several bands, one for each size; the smallest band is farthest from the well.

Running both side by side shows whether the enzyme has cut the DNA.

How to draw an agarose gel

Draw a rectangle for the gel. Write cathode (−) at the top and anode (+) at the bottom.
Draw small slots along the top edge and label them wells.
Number the lanes 1 to 4 under the wells.
In lane 1 draw one band near the well: undigested DNA.
In lanes 2 to 4 draw several bands: larger fragments near the wells, smaller ones lower down: digested DNA.
Draw an arrow from − to + labelled direction of DNA movement, and title the figure Agarose gel electrophoresis.

Reading a gel

Problem

A 5 kb linear DNA is cut by an enzyme that has two sites in it. The fragments are 1 kb, 1.5 kb and 2.5 kb. How many bands form, and in what order do they lie from the well?

Working

Two sites in a linear DNA give 2 + 1 = 3 fragments, so 3 bands.
Check: 1 + 1.5 + 2.5 = 5 kb, the whole molecule.
Larger fragments move the least, so they stay nearest the well.
Order from the well: 2.5 kb, then 1.5 kb, then 1 kb (farthest, towards the anode).

Answer

3 bands: 2.5 kb nearest the well, 1.5 kb in the middle and 1 kb farthest.

In real life

Gel electrophoresis lets forensic labs compare crime-scene DNA with a suspect's and lets doctors check a PCR test. Agarose is purified from agar, the seaweed product sold in India as china grass for desserts.

Exam angle

3 marks: “Explain the principle of agarose gel electrophoresis. How are the separated DNA fragments seen and recovered?” DNA is negatively charged and moves to the anode; agarose sieves fragments by size, smaller ones moving farther. Stain with ethidium bromide and expose to UV: orange bands. Cut out the band and extract the DNA (elution).

2 marks: “Why does DNA move towards the anode in gel electrophoresis? Which fragments move the farthest?” Its phosphate groups make DNA negatively charged, so it moves towards the positive electrode. The smallest fragments move farthest.

1 marks: “Name the stain used to see DNA bands in a gel.” Ethidium bromide, viewed under UV light.

Remember it

DNA is negative, so it runs to the positive pole; the smallest runs farthest.