Genetic Roots

A SCienceLab activity

Dr. Bert Ely Department of Biological Sciences

University of South Carolina715 Sumter St.

Columbia, SC 29208ely@sc.edu

Additional Contributors: Brice Gill, Teresa Pizzuti, Karen Walton, and Jonathon Singer

DNA, a Link to Your Ancestors

Did you know that shortly after George Washington became president, a young woman gave birth to a baby girl and that you have DNA that is identical to some of that baby’s DNA? A few years later, a boy was born in a distant place and his mother worried about whether he would survive. Fortunately, he did because part of the DNA sequence from one of his children is now in your cells. Copies of those DNA segments have passed from parent to child from generation to generation until one of your parents passed them to you! In fact, if that baby was your great, great, great grandmother’s great, great, great grandmother, then she was one approximately 1000 people who were born at that time and contributed to your DNA!

DNA is the basis of life. It contains a set of instructions for building all of the proteins and RNA found in a cell. Those instructions are written in a code called the genetic code. The code consists of 4 bases, Adenine, Cytosine, Guanine, and Thymine, often referred to as A, C, G, and T. The 4 bases are read in groups of three so there are 64 possible combinations (4 possibilities at each of 3 positions). Each combination of three bases forms a code word called a codon. All but three of these codons code for one of the 20 amino acids commonly found in proteins. The order of these codons on the DNA determines the order of the amino acids in the protein that is made from this DNA code. The remaining codons are called stop codons because they tell the cell to stop making a particular protein. A gene contains a set of code words followed by a stop codon. The set of code words show the cell how to make a particular protein. Thus, a gene is a set of instructions for a making a particular protein.

Your DNA is packaged in chromosomes. Each chromosome contains lots of genes so it codes for lots of proteins. You got one set of chromosomes from your mother and a second set from your father. Since, you have two copies of each of your genes, if one copy of a gene happens to contain a mistake in the genetic code, you can use the other copy to make the corresponding protein.

In this exercise, students will isolate their own DNA and amplify a portion of it so that they can see some of the genetic diversity that is present in their class.

UNIT OF STUDY: GENETIC ROOTS

CLASS DESCRIPTION: HIGH SCHOOL

Science Standards addressed:

B-1.1 Generate hypotheses based on credible, accurate, and relevant sources of scientific information.

B-1.2 Use appropriate laboratory apparatuses, technology, and techniques safely and accurately when conducting a scientific investigation.

B-1.6 Evaluate the results of a controlled scientific investigation in terms of whether they refute or verify the hypothesis.

B-1.9 Use appropriate safety procedures when conducting investigations.B-4.2 Summarize the relationship among DNA, genes, and chromosomes.B-4.6 Predict inherited traits by using the principles of Mendelian genetics (including

segregation, independent assortment, and dominance).B-4.7 Summarize the chromosome theory of inheritance and relate that theory to Gregor

Mendel’s principles of genetics. B-4.8 Compare the consequences of mutation in body cells with those in gametes.

Objectives:

Be able to identify the chemical building blocks of DNA. Understand the principles of gel electrophoresis and be able to isolate DNA. Understand how DNA codes for traits. Use DNA analysis techniques to detect genetic diversity.

Laboratory Procedures

Student DNA Sample Isolation

DNA can be obtained from any tissue. To keep the procedure simple, safe and non-invasive, we will use cheek cells. The student simply swabs the inside of his cheek and puts the swab back in the protective tube! No pain. No risk. After the DNA isolation is completed, a portion of the students mitochondrial DNA will be amplified using the PCR technique.

MATERIALS

Sterile buccal swabs (pronounced “buckle”)DNA isolation tubesQuickExtract SolutionMicrofugeWater baths at 65 C and 98 CTubes containing PCR reagents

PROCEDURE

1. Label a microcentrifuge tubes (1.5ml capacity) and add 500µl of QuickExtract DNA Extraction Solution.Note: Be sure to use boil-proof microcentrifuge tubes!

2. Rinse out your mouth with water if it contains food particles.3. Collect tissue by rolling the sample collection swab firmly on the inside of the cheek,

approximately 20 times on each side, making sure to move the brush over the entire cheek.

4. Place the swab end of the collection swab into the microcentrifuge tube containing extraction solution and rotate the swab a minimum of 5 times. While removing the swab from the liquid, make sure to press it against the side of the tube several times to ensure that most of the liquid remains in the tube.

5. Put the cap on the microcentrifuge tube and vortex the mix for 10 seconds. Incubate the tube at 65ºC for 1 minute.

6. Remove the tube from incubation and vortex for 15 seconds.7. Transfer the tube to 98ºC and incubate for 2 minutes.8. Remove and vortex for 15 seconds.9. Pipette 1 ul of your DNA into the PCR tube that matches your sample number.10. Place your PCR tube in the PCR machine.

Agarose Gel Electrophoresis of Food Coloring Dyes

Agarose gel electrophoresis allows you to separate molecules according to size. It is one of the most important procedures used in studies of DNA. To learn how to do it, we will use agarose gel electrophoresis to show that food color dyes are often made up of more than one dye.

Before we start, let's think about what we are going to do. First of all what is agarose? It is a polymer! Poly means many as in polygon (many sides). A polymer is made of many parts. Agarose, a purified form of agar, is a polymer that is made entirely of sugar molecules. It is produced by a kind of seaweed and is used to give thicker consistency to foods such as ice cream. We use agarose because a solution of agarose and water forms a gel when it cools to room temperature. It is sort of like jello except that it does not get soft when it gets warm.

Molecules like food dyes can move through an agarose gel, but the larger they are, the slower they move. To understand the process, think about a backyard or a forest that is full of trees. If you watch, you can see that small birds fly through the branches of the trees almost as if they were not there. What about a large bird like a hawk or an owl? They can only fly through larger spaces among the branches or they have to fly around the trees. Therefore, they cannot fly as fast as the smaller birds. In the same way, small molecules can move quickly among the agarose branches in the gel, but larger molecules move more slowly because they have to pass through larger spaces.

What is electrophoresis? Electrophoresis is process that uses electricity to pull molecules from one place to another. Remember our example above about large vs small birds? How do you think that example relates to gel electrophoresis? If you look at the gel box, you will see that it has two bare wires called electrodes. One is connected to a red wire and has a positive charge, and the other is connected to a black wire and has a negative charge. Most dyes have a negative charge so they are attracted by the positive charge and move through the gel towards the positive electrode. Therefore, we are going to use agarose gel electrophoresis to pull dye molecules through an agarose gel and separate them according to size.

Materials

Food color dyesAgaroseSBA BufferGel apparatusPower supplyPipettesBalanceHeat sourceLiquid measure

Procedure

1) Weigh out 1.2 grams of agarose and add it to 100 ml of room temperature SBA buffer. Swirl to make sure that there are no clumps. Boil the mixture to melt the agarose by heating it in a microwave. As soon as it comes to a boil, open the microwave and swirl the flask without removing it from the microwave. Be sure to handle the hot flask with a glove or hot pad! After swirling, remove the flask and look at the contents. You will see clear particles moving in the solution. These particles are unmelted agarose. Return the flask to the microwave and repeat the boiling and swirling process until you can no longer see the agarose particles. Repeat the boiling and swirling process one more time to be sure all the agarose particles are in solution.

2) Let the agarose solution cool but not too much. It should feel very hot but not so hot that you cannot hold the flask. Pour enough into the gel tray to make a gel that has thickness of about 3 mm. Insert your gel comb into the liquid agarose in the gel tray. Cover the rest of the agarose, let it cool, and save it for your next experiment. (To reuse a solidified agarose solution, simply reheat it with occasional swirling until the solution is uniformly liquid).

3) Once the agarose in your gel tray has solidified, remove the comb. The holes left by the teeth of the comb are called wells. Place the gel tray in the gel box. Add enough SBA buffer to the gel box to cover the gel with about 2 mm of buffer.

4) Slowly and carefully transfer 3 microliters of one of the food dyes into one of the wells. Repeat with the other dyes. (Use every other well of the gel.)

5) Place the lid on the gel box, and turn on the power supply to 200 volts.

6) Look at the gel from time to time to see how the dyes are separating from one another. Notice how each dye moves in a straight line from its well towards the positive electrode. Thus, just like swimmers at a swim meet, each dye stays in its own lane. Also, you can see some dyes moving faster than others. Once the fastest dye moves about two thirds of the way through the gel, turn off the power supply and remove the gel tray from the gel box.

7) For each of the food color dyes, write down the colors you expect to see in the row labeled hypothesis. After electrophoresis, observe which colors are present in each dye, the relative amounts of each color, and the relative distance traveled by each color.

DyeBlue Red Green Yellow

Hypothesis

Observed

DNA Electrophoresis of amplified DNA

Agarose gel electrophoresis can be used to separate DNA molecules according to size. The procedure is the same as when we separated the food dyes. In fact, we load a dye with our DNA samples so that we can monitor the progress of the electrophoresis. The dye solution also contains glycerol to provide a dense mixture that will stay in the bottom of the well.

MATERIALS

DNA samplesDNA size standardLoading dye solutionAgaroseSBA bufferEthidium Bromide solution UV light boxGel box and trayPower supply

PROCEDURE

1. Pour an agarose gel and cover it with SBA buffer as described previously.

2. Spot 1 ul of the loading dye onto a piece of parafilm.

3. Pipette 5 ul of your DNA sample onto the spot, mix by pipetting up and down and then load the mixture into a well of the agarose gel.

4. Pipette 5 ul of a solution containing a DNA size standard into an empty well of the gel.

5. Place the lid on the gel box and turn on the power supply to 200 volts.

6. After the tracking dye has migrated half the way through the gel, turn off the power, remove the gel from the gel box.

7. Observe the DNA by placing the gel on a UV light box. What kind of variation do you see?

DNA EXTRACTION FROM ONION

Prepared by the Office of Biotechnology, Iowa State University

INTRODUCTION

DNA is present in the cells of all living organisms. This procedure is designed to extract DNA from onion in sufficient quantity to be seen and spooled. It is based on the use of household equipment and supplies.

MATERIALS

For teacher preparation

two 4-cup measuring cups (1000 ml) with ml markings one 1-cup measuring cup (250 ml) with ml markings measuring spoons sharp knife for cutting onion large spoon for mixing food processor or blender thermometer that will measure 60o C (140o F), such as a candy thermometer strainer or funnel that will fit in a 4-cup measuring cup cheese cloth (or a coffee filter – takes longer) hot tap water bath (60o C)(a 3-quart saucepan works well to hold the water) ice water bath (a large mixing bowl works well) distilled water light-colored dishwashing liquid or shampoo, such as Dawn or Suave Daily Clarifying

Shampoo large onion table salt, either iodized or non-iodized

Supplies provided to the class

1 test tube for each student that contains the onion solution. Pasteur pipettes or medicine droppers 95% ethanol (grain alcohol) laboratory instructions

TEACHER PREPARATION

1. Set up hot water bath at 55-60o C and an ice water bath.

2. For each onion, add one level 1/4 teaspoon (1.5 g) of table salt to 90 ml of distilled water in a 1-cup measuring cup (250 ml beaker). After the salt is dissolved, add one tablespoon (10 ml) of liquid dishwashing detergent or shampoo and mix gently to avoid foaming.

3. Coarsely chop one large onion with a food processor or blender (may be done by hand if neither is available) and put into a 4-cup measuring cup (1000 ml). For best results, do not chop the onion too finely. The size of the pieces should be like those used in making spaghetti. It is better to have the pieces too large than too small.

4. Cover chopped onion with the 100 ml of solution from step 2. The liquid detergent causes the cell membrane to break down and dissolves the lipids and proteins of the cell by disrupting the bonds that hold the cell membrane together. The detergent causes lipids and proteins to precipitate out of the solution. NaCl enables nucleic acids to precipitate out of an alcohol solution because it shields the negative phosphate end of DNA, causing the DNA strands to come closer together and coalesce.

5. Put the measuring cup in a hot water bath at 55-60o C for 10-12 minutes. During this time, press the chopped onion mixture against the side of the measuring cup with the back of the spoon. (Do not keep the mixture in the hot water bath for more than 15 minutes because the DNA will begin to break down.) The heat treatment softens the phospholipids in the cell membrane and denatures the DNAse enzymes which, if present, would cut the DNA into small fragments so that it could not be extracted.

6. Cool the mixture in an ice water bath for 5 minutes. During this time, press the chopped onion mixture against the side of the measuring cup with the back of the spoon. This step slows the breakdown of DNA.

7. Filter the mixture through four layers of cheese cloth placed in a strainer over a 4-cup measuring cup. When you filter the onion mixture, try to keep the foam from getting into the filtrate. It sometimes filters slowly, so you might want to put the whole set up in the refrigerator and let it filter overnight.

8. Dispense the onion solution into test tubes, one for each student. The test tube should contain about 1 teaspoon of solution or be about 1/3 full. For most uniform results among test tubes, stir the solution frequently when dispensing it into the tubes. There is not an advantage to dispensing more than one teaspoon of solution into a test tube. The solution can be stored in a refrigerator for about a day before it is used for the laboratory exercise. When the solution is removed from the refrigerator, it should be gently mixed before the test tubes are filled.

DNA EXTRACTION FROM ONION

STUDENT INSTRUCTIONS

The process of extracting DNA from a cell is the first step for many laboratory procedures in biotechnology. The scientist must be able to separate DNA from the unwanted substances of the cell gently enough so that the DNA is not broken up.

We have already prepared a solution for you, made of onion treated with salt, distilled water and dishwashing detergent or shampoo. An onion is used because it has a low starch content, which allows the DNA to be seen clearly. The salt shields the negative phosphate ends of DNA, which allows the ends to come closer so the DNA can precipitate out of a cold alcohol solution. The detergent causes the cell membrane to break down by dissolving the lipids and proteins of the cell and disrupting the bonds that hold the cell membrane together. The detergent then forms complexes with these lipids and proteins, causing them to precipitate out of solution.

PROCEDURE

1. Add cold alcohol to the test tube to create an alcohol layer on top of about 1 cm. For best results, the alcohol should be as cold as possible. Slowly pour the alcohol down the inside of the test tube with a Pasteur pipette or medicine dropper. DNA is not soluble in alcohol. When alcohol is added to the mixture, all the components of the mixture, except for DNA, stay in solution while the DNA precipitates out into the alcohol layer.

2. Let the solution sit for 2-3 minutes without disturbing it. It is important not to shake the test tube. You can watch the white DNA precipitate out into the alcohol layer. When good results are obtained, there will be enough DNA to spool on to a Pasteur pipette. DNA has the appearance of white mucus.

DNA Inheritance Game

State Standard – sex cells result in a new combination of genetic information different from either parent.

This game is whimsical and a lot of fun. At the same time, it teaches the concept that physical traits are encoded by genes that are found on chromosomes.

Contents:1 bag per student, containing 10 snap beads (red, blue, green, orange; if other colors are used change the chart below). Alternatively, regular beads can be threaded onto a straightened paper clip.

Goal: Identify 6 bases on a DNA strand (2 amino acids) and use the “secret decoder” to uncover the participant’s eye color and hair color.

Play:1) Hand out one bag to each student.

2) Instruct them to make a row of six beads to represent a chromosome.

3) Explain that it takes 3 bases to code for an amino acid—the building blocks of proteins. Each gene codes for a string of amino acids that make a protein. However, sometimes a change in just one of those amino acids can change a trait. For simplicity, all of our traits are going to be determined by a single amino acid. In this case, we are building proteins for eye color and hair color (Or you can choose whatever characteristic or feature you’d like). The first 3 code for eye color and the second 3 for hair color.

Once they have built their DNA strand, you help them “decode” it. It is the 1st of the 3 that determines the characteristic as shown:

Eye Color Hair ColorRed first: brown redBlue first: blue blackGreen first green blondeOrange first grey orange

Second round

Remove the beads and repeat the process to generate genes for height and skin covering.

Height Skin coveringRed first: tall feathersBlue first: short scalesGreen first medium furOrange first 8 feet slime

Third Round

Remove the beads and repeat the process to generate genes for numbers of toes and limbs.

# toes # limbsRed first: 2 2 legs and 2 wingsBlue first: 1 4 legsGreen first 5 6 legsOrange first 6 8 legs

Ask the students to identify an animal that has each of the characteristics.

Fourth round

Remove the beads and repeat the process to generate genes for height and skin color.

Tail Skin colorRed first: long redBlue first: short yellowGreen first no tail greenOrange first curled purple