DNA gel electrophoresis: separation by fragment size
DNA gel electrophoresis is a technique that separates DNA fragments (pieces of DNA) by size: smaller ones travel further through the gel, a jelly-like mesh of tiny pores.
Remember
DNA carries about the same charge for its mass whatever its length, so charge does not separate the fragments. The gel does, because smaller fragments meet less resistance and travel further.
Memory aid
small runs far, large lags behind, like small people moving through a crowd faster
Picture small people moving through a crowd faster than large ones, just as small DNA fragments pass through the gel more easily and travel further.
DNA is negatively charged, because each phosphate in its phosphate backbone (the repeating sugar-phosphate chain) carries a negative charge. A power supply applies an electric field across the gel, and the DNA is therefore pulled towards the positive electrode, the anode.
A longer fragment has more phosphates and so more charge in total, but it also has more mass. The charge for its mass stays about the same, so the pull on each unit of DNA is about equal and charge does not sort the fragments.
What differs is how easily each fragment passes through the gel, which acts like a sieve. Small fragments slip through quickly and travel furthest, while large fragments are held back and stay nearer the slot where the DNA was loaded. Under fixed conditions, such as the same gel and running time, linear DNA fragments can therefore be separated on the basis of size.
A dye that binds DNA, such as ethidium bromide, makes the DNA visible as bands (the separated fragments). A ladder, a set of fragments of known sizes run alongside, lets you estimate the size of unknown fragments.
The technique is used with restriction enzymes, which cut DNA at specific recognition sites into fragments, and the pattern of band sizes helps identify certain DNA sequences within genes. It is also used after PCR (polymerase chain reaction, which makes many copies of a chosen stretch of DNA): a band at the expected size suggests that the target was amplified (copied many times). A gel shows fragment length, not the base order.
Key facts
- Direction
- DNA is negatively charged and will move towards the anode if placed in a gel electrophoresis electric field.
- Charge per mass
- DNA fragments have about the same amount of charge per mass, so charge does not separate them.
- Order of bands
- in a standard gel, small linear fragments travel furthest, and large ones stay nearer the slot where the DNA was loaded.
- Setup
- the 4 main components are a gel, a buffer, a power supply and a stain or detection dye.
- Using a ladder
- a set of fragments of known sizes run alongside lets you estimate the size of unknown fragments.
- Limit
- a gel shows fragment length, not the base order; DNA sequencing, which determines the order of nucleotides, is needed for that.
Exam traps
- Larger fragments do not migrate faster: a longer fragment does carry more total charge, but it has proportionally more mass, so the charge per unit mass is about the same, and the larger fragment is held back more by the pores of the gel.
- Smaller fragments do not have a higher affinity for the DNA stain: a stain such as ethidium bromide only makes the DNA visible and does not change where it travels.
- DNA moves towards the anode, not the cathode (the negative electrode), because it is negatively charged; the direction is the same for both fragments.
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