Introduction: (Initial Observation)
DNA is a nucleic acid that carries the genetic information in the cell and is capable of self-replication and synthesis of RNA. DNA consists of two long chains of nucleotides twisted into a double helix and joined by hydrogen bonds between the complementary bases adenine and thiamine or cytosine and guanine.
The sequence of nucleotides determines individual hereditary characteristics.
Since DNA holds the genetic information, so the length of DNA molecules and the ratio of DNA to the cell can be different among different species. In this project you will extract DNA from different cells and compare the percentage of DNA by weight.
Information Gathering:
Find out about DNA molecules and the way they keep the genetic information. Read books, magazines or ask professionals who might know in order to learn about DNA extraction and DNA sequencing and extracting segments of DNA. Keep track of where you got your information from.
To understand the methods used in DNA extraction, you need to know about the structure of a cell and how plant cells are different from animal cells. In all DNA extraction methods you will first break the sample to small pieces to make a cell soup. If you are extracting the DNA of blood or the DNA of bacteria in a growth medium, your cell soup is already ready. But for other DNA sources such as banana, you will use a blender, a banana and some water to make your cell soup.
The next step is breaking the cell membrane. Most cell membranes are composed of two layers of lipids or fats as illustrated in the picture above. Each layer is composed of individual lipid molecules that have two distinct regions. The head of the lipid likes to be near water and the tail region does not like water. Detergents are usually used to dissolve or break a part this layer of fat.
Note: Plant cells also have a cell wall made of cellulose. That is why using a blender is necessary in order to break the cell wall.
After adding detergents to your cell soup and stirring it, you may also want to warm it up a little. Up to 60º C is usually safe and will not denature the DNA. Little heat will help breaking the cell wall.
The cell nucleus also have another membrane that will be broken at the same stage. At this time your cell soup is changed to a soup of proteins, plasma, DNA, detergent and fat.
This is a good time to filter the soup to separate any solid material left in the solution and get a clear solution (not very clear, just free from solids).
At this time long DNA molecules are still wrapped around proteins. To separate DNA from proteins different methods are used. Enzymes can do this, however solvents such as Chloroform have also been used to do this. Apparently Chloroform is able to dissolve protein.
Final step is usually a cold solution of alcohol. Cold DNA does not dissolve in cold alcohol, but everything else in the solution does.
Question/ Purpose:
What do you want to find out? Write a statement that describes what you want to do. Use your observations and questions to write the statement.
The purpose of this project is to extract DNA from different plant and animal cells and then compare the percent of DNA among different species.
Identify Variables:
When you think you know what variables may be involved, think about ways to change one at a time. If you change more than one at a time, you will not know what variable is causing your observation. Sometimes variables are linked and work together to cause something. At first, try to choose variables that you think act independently of each other.
Independent variable is the type of organism that we test and measure the percent of DNA (Onion, banana, liver, …)
Dependent variable is the percent of DNA in cell.
Controlled variables are the DNA extraction method and chemicals.
Hypothesis:
Based on your gathered information, make an educated guess about what types of things affect the system you are working with. Identifying variables is necessary before you can make a hypothesis.
My hypothesis is that the percentage of DNA in animal cells is more than the percentage of DNA in plant cells. I also think that more complex organisms have a higher percent of DNA in their cells. So the percent of DNA in bacteria is less than the percent of DNA in fish.
Experiment Design:
Design an experiment to test each hypothesis. Make a step-by-step list of what you will do to answer each question. This list is called an experimental procedure. For an experiment to give answers you can trust, it must have a “control.” A control is an additional experimental trial or run. It is a separate experiment, done exactly like the others. The only difference is that no experimental variables are changed. A control is a neutral “reference point” for comparison that allows you to see what changing a variable does by comparing it to not changing anything. Dependable controls are sometimes very hard to develop. They can be the hardest part of a project. Without a control you cannot be sure that changing the variable causes your observations. A series of experiments that includes a control is called a “controlled experiment.”
Procedure:
First learn and practice DNA extraction so you will be expert on doing that. Then repeat the DNA extraction on different plant and animal cells and measure the ratio of DNA in the cells that you test. Since DNA is the blueprint for life, everything living contains DNA. Tomato, spinach, broccoli, banana, beans and onion can be among the plant cells that you can try to extract their DNA. Beef liver, lamb brain and chicken breast are among the animal cells that you may test. I recommend these samples because they are relatively easier to break down and extract DNA.
How to Extract DNA from plant or animal cells
| To extract DNA we will use:Detergent eNzymes (meat tenderizer) Alcohol |
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Prepare your DNA source in a blender:
- Your DNA source (about 100ml or 2/5 cup of split peas, fresh or frozen)
- A large pinch of table salt (less than 1ml or 1/8 teaspoon)
- Twice as much cold water as the DNA source (about 200ml or 4/5 cup)
Blend on high for 15 seconds.
The blender separates the pea cells from each other, so you now have a really thin pea-cell soup. Because this step is pretty messy, certain sources of DNA should not be used, such as:
And now, those 3 easy steps:
1. Pour your thin pea-cell soup through a strainer into another container (like a measuring cup).
How much pea soup do you have? Add about 1/6 of that amount of liquid Detergent (about 30ml or 2 tablespoons) and swirl to mix. Let the mixture sit for 5-10 minutes.
Pour the mixture into test tubes or other small glass containers, each about 1/3 full.
The basic unit of any living organism. It is a small, watery, compartment filled with chemicals and a complete copy of the organism’s genome.
| Why add detergent?Blending separated the pea cells.
But each cell is surrounded by a sack (the cell membrane). DNA is found inside a second sack (the nucleus) within each cell. To see the DNA, we have to break open these two sacks. We do this with detergent. Why detergent? How does detergent work? Think about why you use soap to wash dishes or your hands. To remove grease and dirt, right? Soap molecules and grease molecules are made of two parts:
Both soap and grease molecules organize themselves in bubbles (spheres) with their heads outside to face the water and their tails inside to hide from the water.
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When soap comes close to grease, their similar structures cause them to combine, forming a greasy soapy ball. A cell’s membranes have two layers of lipid (fat) molecules with proteins going through them. When detergent comes close to the cell, it captures the lipids and proteins. |
2. Add a pinch of enzymes to each test tube and stir gently. Be careful! If you stir too hard, you’ll break up the DNA, making it harder to see.
Use meat tenderizer for enzymes. If you can’t find tenderizer, try using pineapple juice or contact lens cleaning solution.
What is an enzyme?
Enzymes are proteins that help chemical reactions happen more quickly. Without enzymes, our bodies would grind to a halt.
In this experiment, the enzyme we are using comes from meat tenderizer and cuts proteins just like a pair of scissors.
After the detergent step, what do you have in your pea soup?
The cell and nuclear membranes have been broken apart,
as well as all of the organelle membranes,
such as those around the mitochondria and chloroplasts.
So what is left?
- Proteins
- Carbohydrates (sugars)
- DNA
The DNA in the nucleus of the cell is molded, folded, and protected by proteins. The meat tenderizer cuts the proteins away from the DNA.
3. Tilt your test tube and slowly pour rubbing alcohol (70-95% isopropyl or ethyl alcohol) into the tube down the side so that it forms a layer on top of the pea mixture. Pour until you have about the same amount of alcohol in the tube as pea mixture.
DNA will rise into the alcohol layer from the pea layer. You can use a wooden stick or other hook to draw the DNA into the alcohol.
What is that stringy stuff?
Alcohol is less dense than water, so it floats on top. Since two separate layers are formed, all of the grease and the protein that we broke up in the first two steps and the DNA have to decide:
“Hmmm…which layer should I go to?”
This is sort of like looking around the room for the most comfortable seat. Some will choose the couch, others might choose the rocking chair.
In this case, the protein and grease parts find the bottom, watery layer the most comfortable place, while the DNA prefers the top, alcohol layer.
DNA is a long, stringy molecule that likes to clump together.
Protein is a large complex molecule made up of one or more chains of amino acids. Proteins perform a wide variety of activities in the cell.
DNA is a long, stringy molecule that likes to clump together.
Congratulations! You have just completed a DNA extraction!
Now that you’ve successfully extracted DNA from one source, you’re ready to continue with your main experiment. Weigh a new DNA source and perform all the steps to extract DNA. Be careful not to waste or lose any DNA. Separate as much as possible DNA and place it in another container of cold alcohol. Alcohol will separate any additional water molecules attached to the DNA molecules providing us a more accurate measurement of the amount of DNA. Remove the DNA again and place it on a filter paper with known weight. Blow some air on it to evaporate any excess alcohol and then weigh the paper with DNA. subtract the weight of paper to calculate the weight of DNA. Divide the weight of DNA by the total weight of your test sample to calculate the percentage of DNA.
Repeat these steps for every other sample that you test.
The main goal of doing this experiment is to learn how DNA can be extracted and to see the DNA filaments. Don’t worry about the accuracy of your results when you are measuring the ratio of DNA.
The ratio of DNA in the cell depends on the amount of water in the cell, and the amount of water may change from time to time. Water can enter or exit the cell membrane depending on the environmental conditions.
Additional experiments that you can perform in relation to the DNA extraction are:
- Experiment with different soaps and detergents. Do powdered soaps work as well as liquid detergents? How about shampoo or body scrub?
- Experiment with leaving out or changing steps. We’ve told you that you need each step, but is this true? Find out for yourself. Try leaving out a step or changing how much of each ingredient you use.
- Do only living organisms contain DNA? Try extracting DNA from things that you think might not have DNA.
Make Double Helix DNA Model
Making a model is the best way of learning about the elements of a DNA molecule. You can use your model as a separate school project or as an addition to any DNA related science project.
A well made model enhances your display and results a higher level of attention to your presentation.
Introduction:
With over 100,000 different proteins to manufacture, how the heck does our body get it right?
When one thinks of the amount of information the body needs to keep track of, – eye, hair and skin color, protein sequence, toenail size, etc. – it would seem a task for a supercomputer to record all of the necessary information. In essence it is. But not a supercomputer made of silicon wafers and TV screens, rather one made of an intricate biomolecule called DNA.
DNA (deoxyribonucleic acid) is in the family of molecules referred to as nucleic acids. One strand of DNA has a backbone consisting of a polymer of the simple sugar deoxyribose bonded to something called a phosphate unit. Very unimpressively then, the backbone of a strand of
a strand of DNA resembles this:
| sugar-phosphate-sugar-phosphate-sugar-phosphate-sugar-phosphate-… |
What is impressive about DNA is that each sugar molecule in the strand also binds to one of four different nucleotide bases. These bases: Adenine (A), Guanine (G), Cytosine (C) and Thymine (T), are the beginnings of what we will soon see is a molecular alphabet. Each sugar molecule in the DNA strand will bind to one nucleotide base. Thus, as our description of DNA unfolds, we see that a single strand of the molecule looks more like this:
| C | T | G | A | … | ||||
| sugar- | phosphate- | sugar- | phosphate- | sugar- | phosphate- | sugar- | phosphate- | … |
In our model we use light blue balls for Cytosine, light green balls for Guanine, yellow balls for Adenine and Orange balls for Thymine.
Each strand of DNA contains millions or even billions (in the case of human DNA) of nucleotide bases. These bases are arranged in a specific order according to our genetic ancestry. The order of these base units makes up the code for specific characteristics in the body, such as eye color or nose-hair length. Just as we use 26 letters in various sequences to code for the words you are now reading, our body’s DNA uses 4 letters (the 4 nucleotide bases) to code for millions of different characteristics.
Each molecule of DNA is actually made up of 2 strands of DNA cross-linked together. Each nucleotide base in the DNA strand will cross-link (via hydrogen bonds) with a nucleotide base in a second strand of DNA forming a structure that resembles a ladder. These bases cross-link in a very specific order: A will only link with T (and vice-versa), and C will only link with G (and vice-versa). Thus our picture of DNA now looks like this:
| sugar- | phosphate- | sugar- | phosphate- | sugar- | phosphate- | sugar- | phosphate- | … |
| G | A | C | T | … | ||||
| | | | | | | | | | | ||||
| C | T | G | A | … | ||||
| sugar- | phosphate- | sugar- | phosphate- | sugar- | phosphate- | sugar- | phosphate- | … |
Constructing a model of DNA?
Paint all the balls with water based or latex color. Following are the colors that we used in our model.
Yellow is for Adenine (A)- Green is for Guanine (G)
- Blue is for Cytosine (C)
- Orange is for Thymine (T)
- White is for Sugar
- Red is for Phosphate
Use toothpicks to make pairs of Adenine Thymine with sugars on the ends.
Also make pairs of Cytosine Guanine with sugars on the ends.
These pairs form the steps of the ladder in a DNA molecule.
Connect the wood dowels together using wood glue to make a longer wood dowel. It may take a few hours for glue to dry.
Insert the long wood dowel into the base.
Place the first pair on the base and use a wire or string to tie it to the wood dowel.
Insert toothpicks in red balls (phosphates) so that the ball will be centered on the tooth pick. Insert one red ball over each white ball (sugar) and adjust the angles so your DNA model will become double helix.
Mount the second pair over the previous one. Toothpicks from phosphates will enter the sugars of the new pair.
Continue with another set of phosphates and new pairs on top of each other. After a few rows, use another wire or string to tie the last pair to the column (wood dowel).
Continue that until your DNA model is ready.
If you want to separate your DNA model from the base, you will need to use a small amount of wood glue on the ends of toothpicks. If you do this, you can later remove the strings that you used to tie some of the pairs to the column and your DNA model will be removable. For more strength, you may use a very thin wire or string to connect the center of pairs together. If you do this, tie the string to the center of ladder every few steps and make sure that the string is well stretched.
Materials and Equipment:
List of material can be extracted from the experiment section.
To weigh the sample or the extracted DNA you will need a high precision digital or analog scale with at least 0.1g readability.
Small digital scales from MiniScience.com or klk.com can be used for this experiment; however, they need to be calibrated from time to time.
Results of Experiment (Observation):
Experiments are often done in series. A series of experiments can be done by changing one variable a different amount each time. A series of experiments is made up of separate experimental “runs.” During each run you make a measurement of how much the variable affected the system under study. For each run, a different amount of change in the variable is used. This produces a different amount of response in the system. You measure this response, or record data, in a table for this purpose. This is considered “raw data” since it has not been processed or interpreted yet. When raw data gets processed mathematically, for example, it becomes results.
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Calculations:
You will need to calculate the percent of DNA to the cell by dividing the weight of extracted dry DNA by the weight of your initial sample.
Summary of Results:
Summarize what happened. This can be in the form of a table of processed numerical data, or graphs. It could also be a written statement of what occurred during experiments.
It is from calculations using recorded data that tables and graphs are made. Studying tables and graphs, we can see trends that tell us how different variables cause our observations. Based on these trends, we can draw conclusions about the system under study. These conclusions help us confirm or deny our original hypothesis. Often, mathematical equations can be made from graphs. These equations allow us to predict how a change will affect the system without the need to do additional experiments. Advanced levels of experimental science rely heavily on graphical and mathematical analysis of data. At this level, science becomes even more interesting and powerful.
Conclusion:
Using the trends in your experimental data and your experimental observations, try to answer your original questions. Is your hypothesis correct? Now is the time to pull together what happened, and assess the experiments you did.
Related Questions & Answers:
What you have learned may allow you to answer other questions. Many questions are related. Several new questions may have occurred to you while doing experiments. You may now be able to understand or verify things that you discovered when gathering information for the project. Questions lead to more questions, which lead to additional hypothesis that need to be tested.
Frequently Asked Questions
1. I’m pretty sure I’m not seeing DNA. What did I do wrong?
First, check one more time for DNA. Look very closely at the alcohol layer for tiny bubbles. Often, clumps of DNA are loosely attached to the bubbles.
If you are sure you don’t see DNA, then the next step is to make sure that you started with enough DNA in the first place. Many food sources of DNA, such as grapes, also contain a lot of water. If the blended cell soup is too watery, there won’t be enough DNA to see. To fix this, go back to the first step and add less water. The cell soup should be opaque, meaning that you can’t see through it.
Another possible reason for not seeing any DNA is not allowing enough time for each step to complete. Make sure to stir in the detergent for at least five minutes. If the cell and nuclear membranes are still intact, the DNA will be stuck in the bottom layer. Often, if you let the test tube of pea mixture and alcohol sit for 30-60 minutes, DNA will precipitate into the alcohol layer.
2. Why does the DNA clump together?
Single molecules of DNA are long and stringy. Each cell of your body contains six feet of DNA, but it’s only one-millionth of an inch wide. To fit all of this DNA into your cells, it needs to be packed efficiently. To solve this problem, DNA twists tightly and clumps together inside cells. Even when you extract DNA from cells, it still clumps together, though not as much as it would inside the cell.
Imagine this: the human body contains about 100 trillion cells, each of which contains six feet of DNA. If you do the math, you’ll find that our bodies contain more than a billion miles of DNA!
3. Can I use this DNA as a sample for gel electrophoresis?
Yes, but all you will see is a smear. The DNA you have extracted is genomic, meaning that you have the entire collection of DNA from each cell. Unless you cut the DNA with restriction enzymes, it is too long and stringy to move through the pores of the gel; instead, all you will end up seeing is a smear.
4. Isn’t the white, stringy stuff actually a mix of DNA and RNA?
That’s exactly right! The procedure for DNA extraction is really a procedure for nucleic acid extraction. However, much of the RNA is cut by ribonucleases (enzymes that cut RNA) that are released when the cells are broken open.
Possible Errors:
If you did not observe anything different than what happened with your control, the variable you changed may not affect the system you are investigating. If you did not observe a consistent, reproducible trend in your series of experimental runs there may be experimental errors affecting your results. The first thing to check is how you are making your measurements. Is the measurement method questionable or unreliable? Maybe you are reading a scale incorrectly, or maybe the measuring instrument is working erratically.
If you determine that experimental errors are influencing your results, carefully rethink the design of your experiments. Review each step of the procedure to find sources of potential errors. If possible, have a scientist review the procedure with you. Sometimes the designer of an experiment can miss the obvious.
References:
List of References
http://www.sidwell.edu/us/science/vlb5/Labs/DNA_Extraction_Lab/dna_extraction_lab.html
http://www.exploratorium.edu/ti/human_body/dna.html
http://biotech.biology.arizona.edu/labs/DNA_Kiwifruit_teacher.html
Want to learn more?
Here are some books to try:
- DNA Is Here to Stay, by Dr. Fran Balkwill. 1992. ISBN # 0-00-191165-1 (Ages 9-15)
- Amazing Schemes within Your Genes, by Dr. Fran Balkwill. 1993. ISBN # 0-00-196465-1 (Ages 9-15)
- Double Talking Helix Blues, by Joel Herskowitz. 1993. Book and cassette tape. ISBN # 0-87969-431-9 (Ages 8 and up)
- Ingenious Genes, by Patrick A. Baeuerle and Norbert Landa. Barron’s Educational Series, Inc., Hauppauge, NY. 1997. ISBN # 0-7641-5063-4 (Ages 8-12).
- How the Y Makes the Guy, by Norbert Landa and Patrick A. Baeuerle. Barron’s Educational Series, Inc., Hauppauge, NY. 1997. ISBN # 0-7641-5064-2 (Ages 8-12).
