Gel electrophoresis

Gel electrophoresis is a technique that separates DNA fragments (or RNA and proteins) by size. The samples are loaded into wells in a gel and an electric current is passed through it. DNA is negatively charged, so the fragments move through the gel towards the positive electrode, and shorter fragments move faster and further than longer ones. The result is a pattern of bands that shows how long the fragments are.

How does gel electrophoresis work?

Two things make it work: charge and a sieve.

• Charge: every nucleotide in DNA contains a phosphate group, which carries a negative charge. When a current flows, the DNA is attracted to the positive electrode (the anode) and repelled from the negative one.
• The sieve: the gel, usually made of agarose, is a mesh of fibres with tiny pores. Short fragments slip through the pores easily; long fragments get tangled and move more slowly.

Every DNA fragment has about the same charge for each unit of length, so the only thing that changes how fast a fragment moves is its length. After the current has run for a while, fragments of the same length end up together in a band, and the bands are spread out in order of size, with the shortest furthest from the wells.

Gel electrophoresis steps

1. Prepare the DNA. Cut it into fragments with restriction enzymes, or copy the region of interest many times with PCR.
2. Make the gel. Melt agarose in a buffer solution, pour it into a tray with a comb in it, and let it set. Removing the comb leaves a row of wells.
3. Set up the tank. Place the gel in the tank with the wells at the negative electrode end, and cover it with buffer, which conducts the current.
4. Load the samples. Mix each DNA sample with a loading dye, which makes it denser so it sinks into the well and lets you see it moving. Pipette each sample into its own well, and load a DNA ladder into another.
5. Run the gel. Switch on the current. Stop before the fastest fragments run off the far end.
6. See the bands. DNA is colourless, so stain the gel with a dye that binds to DNA and view it, for example under UV light. In DNA profiling, labelled DNA probes can be used to show only the fragments you are interested in.
7. Compare. Compare the bands with the ladder and with each other.

Reading gel electrophoresis results

A DNA ladder (also called a DNA marker) is a mix of fragments of known lengths. Run in its own lane, it acts as a ruler: compare a sample band with the ladder bands beside it to estimate its length in base pairs (bp).

Distance travelled is not proportional to length. Each time the fragment length halves, the band moves on by roughly the same extra distance: in the gel above, 2000, 1000, 500 and 250 bp are evenly spaced. So distance travelled goes down with the logarithm of the length.

Example: estimating a fragment size

In the gel, the ladder bands are 10 mm (2000 bp), 19 mm (1000 bp), 28 mm (500 bp) and 37 mm (250 bp) from the wells. Sample A has one band at 23.5 mm. Estimate its length.

23.5 mm is halfway between the 1000 bp band and the 500 bp band. Because the spacing follows the logarithm of the length, halfway between them is not 750 bp but the geometric mean: √(1000 × 500) ≈ 710 bp. In an exam, "between 500 and 1000 bp" is usually enough unless you are asked for a value; for accuracy, plot distance against log(length) for the ladder and read off the sample.

What is gel electrophoresis used for?

• DNA profiling (genetic fingerprinting). Comparing band patterns from different people for forensic science, paternity testing and studies of how closely related organisms are.
• Checking PCR and genetic engineering. A band of the expected length shows that the right piece of DNA was copied or cut out.
• Medical diagnosis. Spotting an allele linked to a heritable condition, for example when a mutation changes where a restriction enzyme cuts.
• Animal and plant breeding. Checking parentage and avoiding inbreeding.
• Separating proteins. A similar method with a different gel separates proteins by size.

Genetic fingerprinting

Genomes contain many variable number tandem repeats (VNTRs): short sequences repeated one after another, where the number of repeats varies between people. The chance of two people having the same VNTRs is very low. Copy the VNTR regions with PCR, separate them by gel electrophoresis, and each person gives a different pattern of bands.

Each band in a child's profile must come from one parent or the other, because a child inherits one copy of each chromosome from each parent. In a paternity test, any band in the child that is not in the mother must be in the father. At a crime scene, a suspect whose bands all match the evidence sample is very likely to be its source, while a single mismatch rules a suspect out.

Check your understanding.

Common mistakes

• Saying DNA moves to the negative electrode. DNA is negative, so it moves to the positive electrode.
• Saying bigger fragments move further. Smaller fragments move further, because the gel holds back long ones.
• Explaining it by mass alone. The key idea is that fragments are separated by length as they squeeze through the pores of the gel.
• Assuming distance is proportional to length. It goes with the logarithm of the length, so use the ladder bands either side of your band.
• Putting the wells at the positive end. The DNA would run straight out of the gel.

Practice questions

1. Why does DNA move towards the positive electrode?
2. Why is a DNA ladder run in the same gel as the samples?
3. Sample B in the gel above has a band at 32.5 mm. Estimate its length.
4. A child has four bands in a DNA profile. Two match bands in the mother's profile. What can you say about the other two?

Answers: 1) The phosphate groups give DNA a negative charge, and opposite charges attract. 2) Its fragments have known lengths, so it lets you estimate the length of sample fragments run under exactly the same conditions. 3) Halfway between 500 bp and 250 bp, so about √(500 × 250) ≈ 350 bp. 4) They must have come from the biological father, so his profile should contain them.

Practise with a short timed paper

Gene technology, including gel electrophoresis and genetic fingerprinting, is in the second year of A-level Biology. The daily A-level Biology: Year 2 paper is a short timed paper of 8 questions, with a new one every day.

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