How DNA is separated
Gel electrophoresis uses an electric field to move charged molecules through a porous gel. For DNA analysis, an agarose gel is used.
The phosphate groups in DNA give the molecules an overall negative charge. DNA fragments therefore migrate from the end of the gel near the negative electrode towards the positive electrode.
Fragment length affects the rate of movement through the pores in the gel:
- shorter fragments pass through the pores more readily and migrate a greater distance in a given time
- longer fragments encounter more resistance and therefore remain nearer the wells.
Running a DNA gel
- Prepare an agarose gel with wells at the end nearest the negative electrode.
- Place the gel in an electrophoresis tank containing a suitable buffer solution.
- Use a micropipette to load the DNA samples into separate wells.
- Load a DNA ladder or standard into another well so the fragment positions can be compared with fragments of known length.
- Connect the negative electrode at the well end and the positive electrode at the opposite end.
- Apply an electric current and allow the fragments to migrate through the gel.
| Feature | Importance |
|---|---|
| Negative charge of DNA | Determines movement towards the positive electrode. |
| Porous agarose gel | Allows fragments of different lengths to migrate at different rates. |
| DNA ladder or standard | Provides fragments of known length against which sample-band positions can be compared. |
Using enough DNA
A DNA sample must contain enough material for the separated fragments to be analysed. Where the starting quantity is very small, PCR can first be used to amplify the DNA.
Fragment size is inferred by comparing migration with a DNA standard rather than from the migration distance alone.
Exam Tip: Explain both parts of the separation: DNA moves towards the positive electrode because it is negatively charged, while fragments separate because shorter pieces move more readily through the gel.