8.4.3 Investigating the Specificity of Restriction Enzymes

• The specificity of restriction enzymes can be investigated using extracted DNA and gel electrophoresis
• Gel electrophoresis is a technique used widely in the analysis of DNA. During electrophoresis, an electric current is used to separate the DNA molecules according to their size / mass and their net (overall) charge
• The separation occurs because:
  • DNA is negatively charged due to the phosphate groups and so when placed in an electric field the molecules move (migrate) towards the positive electrode
  • Different sized molecules move through the gel at different speeds. The tiny pores in the gel result in smaller molecules moving quickly, whereas larger molecules move slowly

Separation of Restriction Fragments Using Electrophoresis

• The DNA fragments produced by restriction enzymes are known as restriction fragments
  • When a sample of extracted DNA is digested (hydrolysed) by restriction enzymes, a number of restriction fragments of different lengths are produced
  • The number and size of these restriction fragments can be found using gel electrophoresis followed by visualisation of the DNA

• First, a restriction enzyme is added to the sample of extracted DNA, creating a sample of restriction fragments
• Next, these restriction fragments are placed in a well that is cut into the gel nearest to the negative electrode
• An electric current is then passed through the gel, causing the negatively charged restriction fragments to migrate through the pores in the gel towards the positive electrode
  • The restriction fragments move through the gel at different speeds due to their different sizes (lengths)

A cross-section through a gel showing how the restriction fragments migrate through it towards the positive electrode

Visualisation of Restriction Fragments

• As the restriction fragments move through the gel at different speeds due to their different sizes (lengths), bands of restriction fragments are formed in the gel after electrophoresis
• However, DNA is colourless, so the restriction fragments must be treated in such a way so that these bands can be seen
  • This can be done using a stain, resulting in a series of coloured bands in the gel
  • This can also be achieved by treating the DNA with a radioactive marker or adding fluorescent probes that bind to the DNA

• As restriction enzymes are highly specific, they should always produce the same number and sizes of restriction fragments (if the same initial sample of extracted DNA is used)
• This means that an unknown restriction enzyme can be easily identified by comparing the restriction fragments it produces to those of a known restriction enzyme, as the two sets of bands produced by electrophoresis should be exactly the same

Bands of DNA (made up of the restriction fragments) are produced by electrophoresis and observed by staining the DNA. If two sets of the bands match up, then these must have been created by the same restriction enzymes, as these enzymes are highly specific.