Getting the DNA Out and Clean

Recombinant DNA technology begins with a very practical requirement: to cut DNA with restriction enzymes, the DNA must be pure — free from other macromolecules. And DNA does not sit exposed; it is locked away inside cells, wrapped in membranes and surrounded by RNA, proteins, polysaccharides and lipids.

So the first job is to break the cell open and release its DNA along with all these other molecules. The wall is dissolved using enzymes chosen to match the organism: lysozyme for bacteria, cellulase for plant cells, and chitinase for fungi. Each enzyme targets the particular material that makes up that cell's wall.

Stripping Away the Other Molecules

With the cell broken open, the DNA is out but far from pure. Genes lie on long molecules of DNA that are intertwined with proteins such as histones, and the extract is also full of RNA and other material. These have to be removed one by one, each with its own treatment.

The RNA is removed by treatment with ribonuclease (RNase), and the proteins are removed by treatment with protease. Other molecules are cleared away by suitable treatments of their own. What is left, after this stepwise clean-up, is DNA freed from the macromolecules it started out tangled with — ready for the final step of collection.

Precipitating and Spooling the DNA

To bring the purified DNA out of solution, chilled ethanol is added. DNA is not soluble in ethanol, so it precipitates out, appearing as a collection of fine threads suspended in the mixture.

These threads are visible and physically collectable — they can be spooled out by winding them onto a rod or a glass hook. It is a satisfying, tangible end to the isolation process: an invisible molecule made real enough to lift straight out of the tube. At this point we have pure DNA in hand and can move on to cutting it.

Cutting the DNA and Making Recombinant DNA

Cutting is done by restriction-enzyme digestion: the purified DNA is incubated with the restriction enzyme at that enzyme's optimal conditions of temperature and buffer. To follow how far the digestion has gone, we use agarose gel electrophoresis — and because DNA is negatively charged, the fragments move towards the positive electrode, the anode.

Crucially, the vector DNA is cut with the same restriction enzyme, so that its cut ends match those of the fragment. Finally, the cut-out gene of interest and the cut vector are mixed together and joined with DNA ligase. The result of this joining is recombinant DNA — the whole point of the exercise.

Quick Recap

  • To cut DNA with restriction enzymes it must be pure, free of other macromolecules; the cell is broken open using lysozyme (bacteria), cellulase (plant cells) and chitinase (fungi).
  • Genes lie on long DNA molecules intertwined with histone proteins; RNA is removed by ribonuclease (RNase) and proteins by protease.
  • Purified DNA precipitates on adding chilled ethanol, appearing as fine threads that can be spooled out.
  • Cutting is done by incubating purified DNA with the restriction enzyme at its optimal conditions; agarose gel electrophoresis checks progress, with DNA moving to the anode.
  • The vector is cut with the same enzyme; the cut gene of interest and cut vector are then joined with DNA ligase to make recombinant DNA.

Solved Examples — Section 11

Q1. Why must DNA be purified before it can be cut with restriction enzymes?

Answer: Because restriction enzymes need DNA in a pure form, free from other macromolecules such as RNA, proteins, polysaccharides and lipids.

Q2. Which enzymes are used to break open bacterial, plant and fungal cells?

Answer: Lysozyme for bacteria, cellulase for plant cells, and chitinase for fungi.

Q3. How are RNA and proteins removed during DNA isolation?

Answer: RNA is removed by treatment with ribonuclease (RNase), and proteins are removed by treatment with protease.

Q4. How is purified DNA finally made visible and collectable?

Answer: By adding chilled ethanol, which precipitates the DNA as fine threads that can be spooled out.

Q5. Towards which electrode does DNA move during agarose gel electrophoresis, and why?

Answer: Towards the anode (positive electrode), because DNA is negatively charged.

Q6. How is recombinant DNA finally prepared after cutting?

Answer: The source DNA and the vector are cut with the same restriction enzyme, then the cut-out gene of interest and the cut vector are mixed and joined using DNA ligase.