Gel electrophoresis

Separation of DNA.


What is a nuclease?

A nuclease is an enzyme that breaks down phosphodiester bonds that hold DNA molecules together. These bonds are usually very stable, except when they are exposed to this type of enzyme. A nuclease breaks down the phosphodiester bond via a hydrolysis reaction. Nucleases can be further organised into endonuclease and exonuclease.

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Difference between endonuclease and exonuclease:

Endonuclease: These enzymes break down phosphodiester bonds from the "inside part" of DNA.

Exonuclease: These enzymes break down the nucleotides on the "end parts" of DNA.

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What is a restriction endonuclease?

Restriction endonucleases are a type of endonuclease from bacteria that are used to cut DNA.

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Examples of restriction endonuclease:

  • EcoRI
  • HindIII

These restriction endonuclease can recognise sequences of DNA (called palindromic). They cut the DNA at these specific points.

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DNA sequences recognised by endonuclease:

  • XhoI recognises the following:


  • HindIII recognises the following:


Both of these result in sticky ends.

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What is a palindromic DNA sequence?

This is a DNA sequence that consists of the same sequence of nitrogenous bases regardless of which way it is read. (5' to 3' or vice versa).

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What is gel electrophoresis?

This is a technique using gel that allows the separation of DNA fragments based on size and charge. The gel has an electric current running through it, allowing for the separation to occur. The fragments travel from one side of the gel to the other. The larger fragments do not travel as far as the smaller fragments.

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What is agarose made up of?

The agarose gel is made up of polysaccharides and sea weed.

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Factors affecting the migration rate of fragments:

  • Size
  • Formation (i.e. the shape)
  • Charge
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Shape of fragments affecting migration distance:

If 2 DNA fragments are of equal size, the different conformations would affect the migration distance travelled though the agarose gel.

The supercoiled fragments travel the fastest, followed by the linear fragments, followed by nicked fragments.

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