8-4-3-investigating-the-specificity-of-restriction-enzymes

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

• The DNA fragments produced by restriction enzymes are known as restriction fragments

  • When a sample of extracted DNA is digested (hydrolysed) by restriction enzymes, several restriction fragments of different lengths are produced

• The number and size of these restriction fragments can be found using gel electrophoresis:

1. Digest the DNA Sample

   • Add a restriction enzyme to the extracted DNA sample. The enzyme cuts the DNA at specific recognition sites, producing a mixture of restriction fragments of varying lengths

2. Load the Gel

   • Prepare a gel and cut wells at one end (near the negative electrode). Carefully load the DNA restriction fragment mixture into the wells using a micropipette

3. Run the Gel

   • Submerge the gel in an electrophoresis buffer within the gel tank. Apply an electric current. The DNA fragments, which are negatively charged, begin to migrate through the gel toward the positive electrode

4. Separate the Fragments by Size

   • Allow the gel to run for a sufficient period. The DNA fragments move at different speeds:

     • Shorter fragments travel faster and farther through the gel

     • Longer fragments move more slowly,

   • This results in the separation of DNA fragments by size

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 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 the same