What is DNA?
Chromosomes are X-shaped objects found in the nucleus of most cells. They consist of long strands of a substance called deoxyribonucleic acid, or DNA for short. A section of DNA that has the genetic code for making a particular protein is called a gene. Read more about DNA on BBC Bitesize.
Alison played these videos to introduce DNA:
DNA Structure and function, with the Amoeba Sisters
DNA clip from Jurassic Park
The new Edexcel GCSE Biology 9-1 syllabus suggests extracting fruit DNA as one of the core practical activities. However, experts in this subject have warned that often what people get when they think they are extracting fruit DNA is pectin (more details at foot of page). We decided to do a more complex activity involving preparing slides of animal cells using a two-stage staining process, so that we could understand the cell structure and what we would have to break through to extract the DNA, before extracting the DNA.
Slide Preparation
First, we made slides of pig liver cells. We wanted to see what the cells looked like under a microscope before we extracted the DNA.The first step was to prepare a suspension of liver cells in saline.
Each group had a chunk of liver, approximately a teaspoonful .
We chopped the liver finely , then put it in a ziplock plastic bag.
We added approximately 10ml of 'normal saline'. Normal saline is a solution of salt in water, and it has similar concentration to blood and tears: about 0.9% salt. It's also known as 'isotonic solution'. It's used here because it helps to preserve the cells in their natural shape. We wanted to see the cells in good condition, before we tried to break them down to extract their DNA.
The liver was squashed and squeezed in the isotonic saline solution, inside the plastic bag.
We filtered the contents of the bag into a clean beaker.
Using a pipette, we placed one or two drops of the liquid filtrate onto a microscope slide. We placed a cover slip on top of the drop.
Angie examined a slide under the microscope, using the screen display. We could make out individual liver cells in the suspension. Next, a drop of Eosin Y stain was added at one side of the coverslip. The stain was pulled through under the coverslip by blotting gently with paper towel on the opposite side of the coverslip. Now we examined the slide again. The liver cells were stained pink and were much easier to identify on the slide. Next, a drop of Methylene Blue stain was added and pulled through in the same way. Now the cells showed up with greater contrast, and with some blue areas. The nuclei of some cells showed up more clearly.
Eosin Y stains all proteins pink, so it will stain all parts of a cell, including the cytoplasm. Methylene Blue stains acidic structures most strongly, so it stains the nucleus because it contains DNA - deoxyribonucleic acid. You can see in the diagram below that there are various structures inside a cell, but at this stage we are most interested in the cell membrane, which is the 'envelope' containing all the cell structures, and the nucleus, which contains the DNA. The nucleus is enveloped by its own nuclear membrane.
The two-stage staining procedure made the cells much easier to see.
Everyone made a slide, stained it with Methylene Blue and Eosin-Y, and viewed it under the microscope. Everyone was able to identify liver cells in their slide.
Extracting DNA
Having seen these delicate cells in good condition, we now wanted to break down the cell membranes and the nuclear membranes. This process is called lysis and when the cell membranes have been disrupted, the cells are lysed. Cell membranes are made from phospholipids - a type of lipid, or fat. We use detergents to dislodge grease from our hair and from plates, and we can use the same sort of detergents to dislodge lipids from cell membranes. We used shampoo in some samples, and washing-up liquid in others. The active ingredient in these detergents is Sodium Laureth Sulphate, or SLS. A few people have an allergy to SLS, but as long as you don't, it's a very useful substance.
We put our liver suspension in a beaker, together with another chunk of liver, and some key ingredients:
- Saline - some tapwater and a small scoop of salt - measurements are unimportant and we're no longer trying to keep the cells in good condition. In fact, we want to break them up, and adding salt will help to do this and will also help to make the DNA clump together.
- Detergent - a squirt or two.
- Pineapple : a chunk of fresh pineapple supplies protease, an enzyme which breaks down proteins. This helps to break down the proteins surrounding and entwined with the DNA.
The solution was left for at least 5 minutes, to allow the detergent to disrupt the cell and nuclear membranes.
We filtered our solutions into a clean beaker, then poured some of the solution into a test tube.
Ice-cold alcohol was gently tricked down the side of the test tube. We wanted the alcohol to form a layer sitting on top of the liver suspension. Alcohol is less dense than water so it will sit on top of the water and liver mixture unless it is disturbed. We used Isopropanol as it's a cheap alcohol which is relatively safe for this purpose.
Where the alcohol met the suspension, we saw white strands form rapidly. This was a precipitate of DNA. DNA is soluble in water but insoluble in alcohol, so where the solution met the alcohol, the DNA precipitated out. Having the alcohol ice-cold helps this process.
DNA is a polymer, so small units readily bond together to form long chains.
Some people fished out some pieces of DNA for further examination. In our test tubes, once we tried to get it out, it generally formed large clumps a bit like chewing gum. However, if you have a larger beaker and fish it out very carefully, you can wind long strands of it onto a toothpick.
Further Reading:
DNA Your Onions? - University of Reading National Centre for Biotechnology Education. Detailed guidelines on this practical. Notes that common examples using strawberries generally produce pectin rather than DNA. Slightly more readable version at Science In School : Discovering DNA
Our activity was adapted from one in the Illustrated Guide to Home Biology Experiments by Robert Bruce Thompson and Barbara Fritchman Thompson. The full text is available free online.
In the following photos you can see the drops of stain being applied to one side of the coverslip, then 'pulled through' by blotting the opposite side with a paper towel. Sometimes it was necessary to add a drop or two of normal saline to help flush it through, if the stain appeared not to be able to move under the cover slip.
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| Pulling the stain through |
After everyone had seen their cell slides under a microscope, we started the process of extracting DNA:
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| A moment of wonder! |
DNA appears!
Attempting to 'spool' the DNA onto a stick; this proved very difficult when using a test tube!
Every single tube yielded copious quantities of precipitate.
