Genetic Roots
A SCienceLab activity
Dr. Bert Ely
Department of Biological Sciences
University of South Carolina
715 Sumter St.
Columbia, SC 29208
ely@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 of 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:
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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 spits into a cup and DNA is isolated from the cells in the
saliva! No pain. No risk. After the DNA isolation is completed, a portion of the students DNA
will be amplified using the PCR technique.
MATERIALS
Pipettes (100µL and 0.5-10µL) and tips
Saliva cups
Boil-proof 1.5mL tubes with 200µL of 10% Chelex solution
Microcentrifuge
Water bath
PCR Tubes containing PCR reagents (PV92 NEW primers)
Gloves
PROCEDURE
1.
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3.
4.
Rinse out your mouth with water if it contains food particles.
Collect saliva by spitting into a cup (just saliva – no phlegm).
Pipette 100µl of saliva into the 1.5mL tube.
Close the cap on the microcentrifuge tube and vortex the mix for 10 seconds. Incubate
the tube at 98ºC for 20 minutes.
5. Centrifuge the tubes at 2500 x g for 4 minutes.
6. Pipette 2 µl of your DNA into the PCR tube that matches your sample number.
Note: Only supernatant should be used in PCR amplification; avoid pipetting any Chelex
resin (as this will inhibit PCR)
7. Place your PCR tube in the PCR machine (using the program A60).
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 dyes
Agarose
SBA Buffer
Gel apparatus
Power supply
Pipettes
Balance
Heat source
Liquid 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.
Food Color Electrophoresis Chart
Blue
Hypothesis
Observed
Red
Dye
Yellow Green
Brown
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 samples
DNA size standard
Loading dye solution
Agarose
SBA buffer
UV light box
Gel box and tray
Power supply
PROCEDURE
1. Pour an agarose gel and cover it with SBA buffer as described previously.
2. Spot 2 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 or white light box with appropriate filters. What
kind of variation do you see?
DNA EXTRACTION FROM SPLIT PEAS
INTRODUCTION
DNA is present in the cells of all living organisms. This procedure is designed to extract DNA
from split peas in sufficient quantity to be seen and spooled. It is based on the use of household
equipment and supplies.
MATERIALS
For teacher preparation
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4-cup measuring cup (1000 ml) with ml markings
either:
o strainer or funnel that will fit in a 4-cup measuring cup OR
o cheese cloth (or a coffee filter – takes longer) and rubber bands
1 cup measuring cup
1/2 cup measuring cup
1 tbsp. measuring spoon
1/8 tsp. measuring spoon
food processor or blender
light-colored dishwashing liquid or shampoo, such as Dawn or Suave Daily Clarifying
Shampoo
table salt, either iodized or non-iodized
split peas
meat tenderizer
small containers of ice to hold the test tubes
Supplies provided to the class
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1 test tube for each student that contains the split pea solution.
Pasteur pipettes with rubber bulbs or medicine droppers
95% ethanol (grain alcohol)
laboratory instructions
TEACHER PREPARATION
1. Add ½ cup of split peas, 1/8 tsp table salt, and 1 cup of cold water to the blender
2. Blend on high for 15 seconds
3. Set the strainer (or cheese cloth) over the 4-cup measuring cup, and pour the blended
solution into it to strain the solution, and then discard the peas.
4. Add 2 tbsp. of liquid detergent to the strained solution, then swirl to mix
5. Let the mixture sit for 5-10 minutes (alternatively, you can let this solution sit overnight
in a refrigerator).
6. Pour the mixture into the test tubes (about 1/3 full).
DNA EXTRACTION FROM SPLIT PEAS
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 split peas treated with salt, water and
dishwashing detergent or shampoo. Split peas are used because they have 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 a pinch of meat tenderizer to the test tube
2. 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.
3. 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.