What does DNA look like?

Saraswathi Jayanthi and Vinay Singh

All of us have heard about the DNA (Deoxyribonucleic acid), but how many of us have seen it? It may surprise you to know that you can actually see the DNA by a simple extraction process. This article is divided into three parts for easy adaptation into a lesson plan.

• Quiz – warming up activity
• Laboratory activity – DNA extraction
• Questions and discussion

As biology teachers we have seen students showing a lot of curiosity and enthusiasm to learn about the DNA – the wonder molecule. Students of middle school are familiar with terms such as prokaryotic cell, eukaryotic cell, nucleus, chromosome, nucleic acids, RNA, and DNA. Though students have an idea about these terms, sometimes they may not be clear as to what these terms actually mean. Before going into a discussion on the structure of the DNA and its role and significance, the teacher can have a small warm up activity like a quiz in class. Here are some questions you can ask your students.
1. What is the difference between a prokaryotic and a eukaryotic cell?
2. Give examples of prokaryotic and eukaryotic cells.
3. What is the key cell organelle in a eukaryotic cell?
4. What is present inside the nucleus?
5. What are nucleic acids?
6. Where do you find them?
7. What do you understand by the term genetic material?
8. What is the difference between DNA and RNA?
9. What is a chromosome?
10. What is a gene?

Guide your students in understanding the terms chromosome, DNA, and gene and sum up the topic as below.
In eukaryotes most of the DNA is held inside the nucleus, though some DNA is contained in the mitochondria and chloroplast. In prokaryotes, the DNA lies in the cytoplasm as a coiled circular molecule known as nucleoid. DNA is the genetic material in most organisms with the exception of a few viruses. It is a negatively charged molecule made up of nucleotides.

Now, as the students have a basic understanding of the DNA, you can have a hands-on activity which enables them to actually see the DNA. Here we present a simple method for the extraction/isolation of the DNA from two sources.

Extraction of DNA from eukaryotic cells – banana
Materials required

• Fresh banana
• Knife
• Mortar with pestle
• Extraction solution/buffer
• 500 ml beaker
• Funnel
• Cheese cloth (cut to fit over a small beaker)
• Test tubes
• Hot water bath set at a constant 60°C or a beaker of water set at 60°C using a thermometer
• Ice water bath (a beaker containing crushed ice)
• Measuring cylinder
• Dropper
• Alcohol
• *Centrifuge (optional)
• Distilled water

Necessary preparations before starting the experiment
1. Extraction solution
• 10 ml of clear shampoo
• 1.5 g of table salt (sodium chloride)
• Distilled water
Add 1.5 g of salt (approximately half a tea spoon) to 90 ml of distilled water and mix well. Add 10 ml of shampoo and stir slowly to avoid foaming of the shampoo.

2. Ice cold 95% alcohol
Measure about 95 ml of absolute alcohol into a reagent bottle and add 5 ml of distilled water. Close the lid of the reagent bottle tightly and keep it in the ice bath or place it in the refrigerator (4°C) for chilling before use.

3. Ice bath: Keep crushed ice in a beaker or a small container.

Procedure of extraction
1. Peel the banana, cut a small piece (one inch/weight approximately 10 grams.) Transfer it into the mortar.
2. Add 10 ml of extraction solution and gently grind to make a paste. Add another 10 ml of extraction solution and mix well.
3. Filter the contents of the mortar through the cheese cloth using a funnel.
4. Gently squeeze the extract and collect the filtrate in a clean test tube.
5. Now place the test tube in the water bath for 10- 15 minutes set at 60- 65°C. The solution becomes clearer after heating.
6. After heating, immediately chill the test tube by keeping it in the ice bath for a few minutes.
7. Centrifuge at 3000 rpm for 10 minutes. Collect the supernatant (clear top solution.) If centrifuge is not available you can proceed to the next step directly.
8. Transfer 4-5 ml of the supernatant or extract into a test tube.
9. Now add an equal amount of ice cold 95% alcohol slowly along the sides of the test tube with a dropper.
10. Keep the tube in ice bath for 10 to 15 minutes.

You will be excited to see DNA in the test tube – the slimy, silky thread- like structure floating in the upper layer of the test tube. Collect the upper alcohol layer along with the DNA into another test tube or dip a glass rod, rotate, and spool out. You can centrifuge it again at 3000 rpm for 5 minutes to precipitate the the DNA. Add 2 ml of distilled water and store it. The extracted DNA can be stored at room temperature for a few days. If you want to keep it for a longer period, store it in the fridge.

You can extract DNA using the above procedure from any other fruit as well. You can plan a laboratory activity by giving different fruits to different groups and ask students to compare their findings.

If you are interested and want to make sure that what you have isolated is the DNA, there is a simple confirmatory test. This is optional and you can try this if you have DPA (Diphenyl amine reagent).

Confirmatory test for the presence of DNA
• Take 2 ml of your DNA sample into a clean test tube.
• Add 4 ml of DPA reagent and mix gently.
• Set a control with distilled water (2 ml) and DPA reagent (4 ml).
• Place the tubes in boiling water bath for 10-15 minutes.
• Cool the tube to room temperature.
• Appearance of bluish colour confirms the presence of DNA in your sample test tube.

Preparation of Diphenylamine Reagent (DPA): Dissolve 1g of pure DPA in 2.5 ml of concentrated sulphuric acid. Make up the volume to 100 ml with glacial acetic acid. This solution must be prepared fresh.

Questions on procedure (Read Tell me why? at the end of the article)
After the experiment you can ask students to research and find answers to the following questions.
• Why do we select fruits like banana/strawberry?
• Why do we grind/mash the fruit?
• Why do we use shampoo in this experiment?
• What does salt do?
• What is the purpose of heating the extract at 60ºC?
• Why do you need to centrifuge the extract?
• What is the purpose of the ice bath?
• What is the role of alcohol?
• Why is ice cold alcohol used?

Laboratory activity 2: Extraction of DNA from prokaryotic cells – Bacteria
In this lab activity you can show the method of isolating the DNA from prokaryotic cells (bacterial). Instead of culturing bacteria on a microbiological media, we have chosen a simple way to obtain bacterial cells.

Materials required
• Potato big 1
• Sugar – a teaspoon
• Knife
• Beaker 500 ml
• Bunsen burner/hot plate
• Strainer
• Test tubes
• Glass rod
• Extraction solution
• Water bath set at 60ºC
• Alcohol
• Ice water bath (a beaker containing crushed ice)
• Test tubes
• Dropper
• Soil solution
• pH paper
• 1 N Sodium hydroxide solution
• Table top centrifuge
• Glass rod
• Distilled water

Procedure of extraction
1. Wash, peel, and cut potato into pieces and boil in 200 ml water till the pieces become soft.
2. Collect the broth by straining.
3. Allow the broth to cool to room temperature and add a spoon of sugar.
4. Check the pH using pH paper. Adjust the pH to around 7 using 1N NaOH solution.
5. Take a little amount of soil in a test tube, add water, and shake well.
6. Add a few drops of soil solution to the potato broth and stir well. Leave the beaker at room temperature for about 24 hours.
7. You can observe haziness in the broth which indicates bacterial growth.
8. Centrifuge the broth at 3000 rpm for 15 to 20 minutes.
9. Collect the pellet (bacterial cells)
10. Suspend the pellet in 2 ml distilled water.
11. Add 8 ml of extraction solution and mix well.
Note: If centrifuge is not available you can use potato broth directly.

Now repeat steps 5 to10 from the procedure of extraction of the DNA from the banana. You will observe the thin, slimy thread-like DNA. But the amount of DNA will be less, as the amount of bacterial DNA is less compared to eukaryotic cells. Also we have not used any microbial media to culture bacteria, so the bacterial cells collected are fewer.

Safety concerns for culturing bacteria: As we are not isolating and handling bacterial cultures, it is not a matter of concern. During the process, we are adding extraction buffer and heating, so bacteria get killed. The unused broth can be discarded after heating or by adding any disinfectant to kill bacteria.

Clean up
After the experiment you need to discard the left over banana extract, potato broth and solutions and clean the glassware and workbench. Close the lid of alcohol and store it in a safe place.

Discussion and conclusion

• What are the various ways in which you can modify the above experiments? As you are aware of the purpose of each ingredient and every step, you can modify the procedure according to the availability of the materials (E.g.: you can put the banana piece in a zip lock bag and squeeze instead of using a mortar and pestle).
• Seeing DNA, the wonder molecule is definitely an exciting experience for children. Apart from the fun and thrill what is the purpose?
  Scientists isolate DNA using sophisticated procedures. This can be used for various purposes like molecular biology experiments, to isolate genes and modify through genetic engineering experiments, for forensic science, comparing DNA of different organisms and so on.

References
Find the DNA in a Banana: Bring Science Home: Activity 9 by Molly Josephs, Scientific American (May 12, 2011).
http://www.scientifi camerican.com/article/fi nd-the-dna-in-abanana-bring-science-home/

Classroom Activities in Plant Biotechnology by Stephens J. and J. Leach, The Plant Health Instructor.
http://www.apsnet.org/edcenter/K-12/TeachersGuide/PlantBiotechnology/Pages/Activity1.aspx

Estimation of DNA using Diphenylamine Method.
http://nptel.ac.in/courses/102103047/6

Tell me why?

1. Why do we select fruits like banana/strawberry?
As all living cells contain DNA, we can extract DNA from any sample like onion, apple, strawberry, kiwi, etc. We chose banana as it is readily available.

2. Why do we grind/mash the fruit?
We need to disorganize the tissue in order to get individual cells so that the extraction solution can act upon the cell membranes to break open the cell boundaries.

3. Why use shampoo?
The cell membrane contains lipids and proteins. Shampoo disrupts the cell membrane and nuclear membrane and releases its contents. We can use any detergent like dish washing liquid. The function of the detergent is to remove the greasy substance from the membranes.

4. What does salt do?
DNA is negatively charged. Salt neutralizes the charge on DNA molecules and makes them stick together. It helps proteins and carbohydrates to precipitate.

5. Why heat the extract at 60ºC?
The temperature is intended to denature proteins, which will otherwise break up the DNA into small fragments. At 60ºC, DNA is safe and no damage occurs. Be careful and do not raise the temperature above 65ºC.

6. Why centrifuge the extract?
This step is optional. By centrifuging, cell debris gets separated and forms a pellet which can be removed.

7. What does ethanol do?
Ethanol removes water from the DNA and precipitates it to form visible white slimy strands.

8. Why use ice bath and ice cold alcohol?
Cells contain enzymes called restriction enzymes and DNases. The function of these enzymes is to destroy the DNA of invading viruses. As the cell DNA is contained in its nucleus it is safe from these enzymes. Once the DNA is released from its nucleus, after detergent action it can be damaged by these enzymes. Under very cold conditions their activity ceases and they cannot act and destroy the DNA being isolated.

Tell me why-1?

1. Why potato?
Any vegetable extract can be used but potato is better as its starch content is more and it is readily available.

2. Why did we add sugar?
By adding sugar we are providing additional sugar for bacterial growth.

3. Why adjust pH to 7?
In potato broth we can expect both fungal and bacterial growth. Fungi grow in acidic pH and neutral pH is ideal for bacterial growth. Fungi grow slower than bacteria; they require at least a couple of days. So we are sure of culturing bacteria without fungal contamination.

4. Why did we add the soil solution?
Bacteria are present everywhere: in soil, water, and even air. Soil solution is added only to enrich/enhance bacterial growth.

Dr. Saraswathi Jayanthi is a biology teacher and ICSE & ISC Coordinator at Sreenidhi International School, Hyderabad. She can be reached at saraswathij@sis.edu.in.

Mr. Vinay Singh is a biology teacher at Sreenidhi International School, Hyderabad. He can be reached at vinays@ sis.edu.in.