Isolating the DNA, cutting it and reading the gel

The steps in order

Making a recombinant DNA is like building a model from a kit. You must do the steps in the right order. Here they are, and the rest of this course follows them.

1. Isolate the DNA from the cells.
2. Cut it with restriction enzymes, and cut the vector too.
3. Separate the fragments and isolate the one you want.
4. Make many copies of the gene, by PCR.
5. Join the gene to the vector with ligase.
6. Put the recombinant DNA into a host.
7. Find the cells that took it up, and the cells with the gene inside.
8. Get the product (the next course).

Step 1: isolating the DNA

DNA lies inside the cell, wrapped by a membrane and, in most cells, a wall. It is mixed with RNA, proteins, lipids and sugars. We must free it and clean it. This is what is done.

• Break the cells open. The enzyme depends on the cell. Lysozyme breaks the wall of a bacterium. Cellulase breaks the wall of a plant cell. Chitinase breaks the wall of a fungus. An animal cell has no wall, so it opens more easily.
• Remove the RNA. The enzyme ribonuclease breaks down RNA.
• Remove the proteins. The enzyme protease breaks down proteins. Other molecules are removed by suitable treatments.
• Make the DNA appear. When chilled ethanol is added, the purified DNA comes out of the solution. It can be seen as a mass of fine threads. It can be wound up on a glass rod, a process called spooling.

Step 2: cutting with restriction enzymes

The pure DNA is now mixed with a restriction enzyme, under conditions in which the enzyme works best. The enzyme cuts the DNA at every one of its sites. The vector is cut with the same enzyme, so that its sticky ends fit the ends of the pieces of the source DNA.

How do we know that the cutting is done? We look at the product. For this we need the technique of gel electrophoresis.

Step 3: gel electrophoresis

This is the standard way to separate DNA fragments by size. Think of a crowd of people running through a forest. The tall ones get stuck on the trees, and the small ones get through quickly. In the gel, the long DNA molecules are slowed down more than the short ones.

• The gel. It is made from agarose, a substance that comes from sea weeds. It forms a mesh, like a sieve. The DNA is placed in small slots, the wells, at one end.
• The charge. DNA has a negative charge, because of its phosphate groups. In an electric field, it moves towards the positive electrode, the anode.
• The sieving. The fragments separate by size. The smaller the fragment, the farther it moves. The distance is nearly a straight line against the log of the size.

• Seeing the bands. DNA cannot be seen in visible light. The gel is stained with ethidium bromide, and then it is placed under UV light. The DNA shows as bright orange bands. Ethidium bromide is a hazard to health, so gloves are used, and safer stains are now common.
• Elution. The band with the wanted fragment is cut out of the gel with a clean blade. The DNA is then taken out of the gel piece. This step is called elution.

Always run a ladder next to the sample. It is a mix of fragments of known sizes. By comparing a band with the ladder, you can tell its size.

You should see that the distance falls as the size grows, and that doubling the time doubles the distance. A fragment that is ten times longer lies about 14 mm nearer to the well, in this model, after 60 minutes.

Try it in Python

The program gives the distance run by each fragment of a digest. The numbers are a model with round values.

Practise with new numbers

Common mistakes

• Saying that DNA moves to the negative electrode. DNA is negative and moves to the positive electrode, the anode.
• Saying that big fragments run farthest. The small ones run farthest.
• Using the wrong enzyme for the wall. Lysozyme for bacteria, cellulase for plants, chitinase for fungi.
• Saying that the ethanol cuts the DNA. It only makes the DNA come out of the solution.
• Forgetting the stain. Without ethidium bromide and UV light, the bands cannot be seen.

Remember these

• The enzymes that open the cells: lysozyme (bacteria), cellulase (plants), chitinase (fungi). Ribonuclease removes the RNA. Protease removes the proteins. Chilled ethanol makes the DNA come out as threads.
• Gel electrophoresis: agarose gel, DNA moves to the anode, smaller fragments move farther.
• Ethidium bromide and UV light show orange bands. The wanted band is cut out and eluted.

Check yourself

Try each one on your own first. If you are stuck, open a hint. Then check with Show the answer.

1. Which enzyme would you use to open a plant cell, and which for a fungus?

2. How is the RNA removed from the preparation, and how are the proteins removed?

3. In which direction does DNA move in a gel, and why?

4. A digest gives fragments of 2000, 800 and 300 bp. Which band lies nearest to the well?

5. How are the bands of DNA made visible, and how is a band taken out of the gel?

References

• NCERT (2023). Biology, Textbook for Class XII, chapter 9, Biotechnology: Principles and Processes.
• Clark M. A., Douglas M. and Choi J. (2018). Biology 2e, OpenStax, chapter 17.2 (Basic techniques to manipulate genetic material).