Isolation of the Genetic Material (DNA)

Recombinant DNA technology involves a set of steps in a specific sequence. The first is to obtain the DNA in a pure form, free from other macromolecules.

  • DNA is enclosed within membranes, so the cells must first be broken open to release the DNA along with other macromolecules. This is done with enzymes that digest the cell wall: lysozyme (for bacteria), cellulase (for plant cells) and chitinase (for fungi).
  • The other macromolecules are then removed with the appropriate enzymes: RNA is removed by ribonuclease (RNase) and proteins by protease.
  • Finally, the purified DNA is precipitated out by adding chilled ethanol, where it appears as a collection of fine threads that can be spooled out.

Recombinant DNA workflow: isolate DNA, cut, ligate into vector, transform host, culture, extract product

One-liners: break cell walls with lysozyme (bacteria) / cellulase (plant) / chitinase (fungus); remove RNA with RNase, protein with protease; DNA precipitates with chilled ethanol (fine threads).

Cutting DNA and Separating the Fragments

Cutting DNA at specific locations

The purified DNA (and the vector) is incubated with a restriction enzyme at its optimal conditions. The enzyme cuts the DNA into fragments; the progress of the digestion is checked by agarose gel electrophoresis.

Gel electrophoresis - separating the fragments

The cut fragments are separated by agarose gel electrophoresis:

  • The agarose gel (a polymer from sea-weeds) is the matrix. Because DNA is negatively charged, when an electric field is applied the fragments move towards the positive electrode (anode).
  • The fragments separate according to size: smaller fragments move farther through the gel than larger ones.
  • The separated DNA is made visible by staining with ethidium bromide and exposing the gel to UV light, where the DNA shows up as bright orange bands.
  • The desired band is cut out from the gel and the DNA extracted - a step called elution. These purified fragments are then used to build the recombinant DNA by joining them to cloning vectors.

One-liners: cut with restriction enzyme; separate by agarose gel electrophoresis - DNA is negative, moves to the anode (+), smaller = farther; stain with ethidium bromide + UV = orange bands; recover the band by elution.