6.3.6 Electrophoresis

Question 1

What does gel electrophoresis do?

Answer 1

• Separates molecules of DNA, RNA or Proteins by their mass and charge
• Allows these molecules to be analysed and for an organism’s genome to be sequenced for GENETIC PROFILING (fingerprinting)

Question 2

What factors does the separation of DNA / RNA / Protein molecules depend on?

Answer 2

• Charge of molecule (positive cations move to cathode, negative anions move to anode. DNA is negatively charged due to phosphate groups so moves towards anode)
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• Sizes of DNA molecules (smaller molecules move quicker and further due to being able to fit through the tiny pores in the gel easier)
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• Type of gel (different gels have different sized pores, affecting the speed which molecules can move through them)

Question 3

What are VNTRs?

Answer 3

• Variable number tandem repeats
• Contain variable numbers of repeated DNA sequences
• Vary between people (except identical twins)
• Restriction endonucleases cut close to these VNTRs
• Fragmenting DNA

Question 4

What are probes?

Answer 4

• Single stranded DNA sequences
• Complementary to CNTR regions
• Contain radioactive labels, causing them to emit radiation making X-ray films dark
• Contain fluorescent dyes / stains which shine in UV light, exposing them

Question 5

Method for Gel Electrophoresis

Answer 5

1. Create agarose gel with wells cut at one end
2. Submerge gel in electrolyte (conducts electricity)
3. Load fragments into wells using micropipette
4. Apply electrical current, anode at opposite end of wells side (negative DNA fragments move towards anode because of the electrostatic attraction)
5. Smaller / shorter DNA fragments move faster from wells than larger / longer fragments
6. Fragments not visible so transferred onto absorbent paper
7. Heated to separate 2 DNA strands
8. Probes added, X-ray or UV light used to reveal pattern of bands revealing the distance different fragments of DNA have travelled across the gel

Question 6

Why might a buffer solution be used for separating proteins?

Answer 6

• Charge of R groups of amino acids depends on pH
• So buffer solutions keep pH constant

Question 7

How are proteins prepared for electrophoresis? (short)

Answer 7

• Denatured (breaking disulphide bonds)
• Protein manipulated into negatively charged rod shape
• To allow separation by size