Electrophoresis
Laura Armstrong & Joe Wolfensohn
Teachers


Contents
Recall Questions
This topic requires prior knowledge of DNA structure. You can test your knowledge on this below.
Which bases pair together in DNA?
Adenine with Thymine, and Cytosine with Guanine.
What charge does a DNA molecule carry?
A negative charge due to its phosphate backbone.
What bond holds complementary DNA bases together?
Hydrogen bonds.
Topic Explainer Video
Check out this @JoeDoesBiology video that explains electrophoresis or read the full notes below. Once you've gone through the whole note, try out the practice questions!
What is Gel Electrophoresis?
Gel electrophoresis is a laboratory technique used to separate DNA fragments, RNA, or proteins based on their mass and charge. It is a key method used in:
- Genetic fingerprinting
- Gene sequencing
- Forensics
- Disease diagnostics
- Protein analysis
How Gel Electrophoresis Works (DNA)
Step-by-Step:
- DNA is cut into fragments using restriction endonucleases (restriction enzymes).
- These DNA fragments are loaded into wells in a slab of agarose gel.
- An electric current is applied across the gel.
- Because DNA is negatively charged, it moves towards the positive electrode (anode).
- The gel acts as a sieve:
-
- Smaller fragments move faster and further.
- Larger fragments move more slowly and travel less distance.
The separated fragments can be visualised by staining the DNA with a loading dye prior to putting the DNA into the wells. (otherwise DNA would not be visible).
The DNA can also be visualised by adding fluorescently labelled probes that bind to the DNA after separation and then using UV light to visualise them.
How Does It Separate DNA Fragments?
- DNA fragments of different lengths will migrate through the gel at different rates.
- DNA fragments with a greater number of base pairs will have a longer length and therefore a greater mass.
- These fragments would not travel as far in the gel compared to DNA fragments with a smaller mass.
- This separation based on mass creates distinct bands, where each band represents a fragment of a particular size.
- A DNA ladder is where DNA fragments of a known length are separated on the gel alongside samples of unknown length.
- The fragments of unknown length can be compared to the fragments of the DNA ladder to estimate their length by comparison.
Can It Be Used for Proteins?
Yes - but the process is slightly different:
- Polyacrylamide gel (not agarose) is often used for higher resolution.
- The proteins will be separated by mass, but this depends on the number of amino acids in the polypeptide chains.
- They are also separated by charge, but this depends on the amino acid R-groups they contain.
Key Terms
- Gel Electrophoresis: A technique used to separate DNA, RNA, or proteins based on mass and charge.
- Restriction Endonuclease: Enzyme that cuts DNA at specific recognition sites.
- DNA Ladder: A mixture of DNA fragments of known lengths used as a size reference.
- Agarose Gel: A gel matrix used to separate DNA/RNA in electrophoresis.
Exam Tip
In exam answers:
- Always mention charge when explaining why DNA moves.
- Always mention DNA fragments are separated by mass / length and remember that smaller fragments move further.
Scientists used DNA probes and electrophoresis to screen four volunteers for five different viral DNA fragments. Lane 1 enabled the size of the different viral fragments to be determined.
Suggest and explain how. (2 marks)
- Lane 1 has DNA fragments of known mass/lengths
- Compare their position to the position of unknown fragments
The lengths of the viral DNA fragments were:
- 650 base pairs
- 300 base pairs
- 585 base pairs
- 350 base pairs
- 550 base pairs.
Which volunteers had at least one of the viral DNA fragments with 300 base pairs or 585 base pairs? (1 mark)
- 3, 5 and 4
- Explanation: 300 base pairs was the shortest length fragment and so would have moved the furthest in the gel.
- 585 base pairs was the second longest fragment and so would have moved the second shortest distance.
Practice Question 1
Try to answer the practice question from the TikTok on your own, then watch the video to see how well you did!
Practice Question 2
Try to answer the practice question from the TikTok on your own, then watch the video to see how well you did!