Fo Sci 14
Isolation of DNA from Human Buccal Cells
Prior to PCR and Gel Electrophoresis
Name _____________________________
Objective:
The purpose of this lab is to treat human buccal (mouth) “cheek” cells with a variety of materials in order to
break open the plasma membrane and the nuclear envelope, releasing the 46 chromosomes from each cell into
solution. The isolated DNA can then be used in DNA fingerprinting. This procedure is very similar to that used
in Forensic labs prior to DNA fingerprinting.
Many thousands of buccal cells will be collected by washing out your mouth. The saliva you collect will contain
1000’s of buccal cells washed off from the sides of your mouth. The DNA that you will see or visualize will be
composed of thousands of strands! You will be able to see a cloud of combined strands of DNA.
Keep in mind that a single DNA molecule is too small to see and it has no color. You will be looking at
thousands of strands together that reflect white light so that you can see a cloudy mass. You will NOT be able
to see the nucleotide sequences that might show differences between people or between different species of
organisms. Those difference are too small to see!
Introduction:
Different procedures can be used to help isolate the DNA from cells. During
isolation, the cell is broken open and the proteins within the cell are separated
from the DNA. We will use salt, detergent and alcohol to break open the cell
and separate the DNA from the proteins that surround it. Heat can also be used
but we will not be using it in our method.
DNA
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Salt: Adding salt to cells will help to break down the cell membrane by
denaturing the proteins in the membrane. This will then release the
DNA. Remember that salt (NaCl) is an ionic compound that dissociates in an aqueous solution to form
Na+ and Cl-. The individual ions will interact with and disrupt hydrogen bonds and other R-group
interactions between amino acids in the proteins. This disrupts the shape of the proteins in the plasma
membrane and the nuclear membrane. The membrane then breaks open, releasing the cytoplasm and the
DNA in the nucleus of the cell. In addition, proteins that are associated with the DNA (like histones) are
denatured and separated from the DNA.
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Liquid detergent: The detergent will act to dissolve or separate the phospholipid components of the cell
membrane. The DNA will be released from the cell. Detergent molecules are similar in structure to
phospholipids. They have both hydrophobic and hydrophilic regions. The similar regions of both
detergent and phospholipids interact with each other, breaking the phospholipids apart and breaking
down the cell membrane. This releases the contents of the cell. The process is similar to the
emulsification of fats by bile in the intestine. The detergent works in a similar way when it breaks apart
the lipid molecules in dirt and grease, so they can more easily be washed away when cleaning dishes or
clothes.
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Cold, 95% ethanol: DNA is soluble in water but not very soluble in alcohol (and even less so when the
alcohol is cold). DNA molecules at the surface of the soapy mixture will tick together as they rise into
the alcohol solution (the densities of the alcohol and DNA are approximately equal). This forms a
whitish, visible cloud of DNA that you can see. This can be collected to be used to form a DNA
fingerprint.
You will be treating the cheek cells with salt, liquid detergent and ethanol in order to isolate the DNA. The
material you will get will be white, slimy and gathered from thousands of cells. Although you won’t be able to
put it under the microscope to observe the double helix (it is much too small to see) you will be able to collect and
hold DNA!
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Materials:
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Plastic test tube
Glass pipettes with pipette pump
8% sodium chloride solution
beaker to hold test tube
Gatorade, graduated cylinders
bathroom drinking cup
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10 % sodium lauryl sulfate solution (dish detergent)
plastic wrap or wax Parafilm film
small, plastic pipette to collect DNA
Alcohol (95% ethyl or isopropyl)-chilled
1-3 dram, screw-top vial
Directions: All the materials used in this experiment can be purchased at a grocery store. This is something you
CAN do at home! The directions for this lab are extremely easy. Use the following procedure to ensure success.
1) Add 0.8 ml sodium chloride solution to the plastic test tube. Set the tube in a beaker.
2) Using a clean pipette, add 4 ml of Gatorade into a clean bathroom cup.
3) Pour the Gatorade into your mouth. Swirl the Gatorade around for at least two minutes. The more
active the swirling, the better. You can also bite on the inside of your mouth to get more cells.
4) Spit the Gatorade back into the drinking cup.
RELEASE THE DNA FROM INSIDE THE CHEEK CELLS:
5) Pour the buccal cell solution into the test tube that contains the salt solution.
6) Add 0.8 ml of the 10% sodium lauryl sulfate solution to the mixture in the test tube.
7) Place plastic wrap over the top of the test tube and mix the contents by GENTLY INVERTING THE
TEST TUBE. Do not shake the test tube!
PRECIPITATE THE DNA:
8) Hold the test tube at a slight angle, carefully add alcohol down the side of the tube so it forms a layer
over the buccal cell mixture.
9) Place the test tube in the beaker to sit for a few minutes. Observe what happens at the interface
between the ethyl alcohol and the buccal cell solution. The clouds of white strands you are
observing are the DNA!
COLLECT THE DNA:
11) Carefully use a clean toothpick to collect the DNA. Transfer some of it to one of the small, alcohol
filled vials for storage. Cap your DNA tightly and it should last a long time.
SAFETY CLEANUP: Toss anything that came in contact with your mouth or the buccal
solution into the trash! Toss the plastic test tube, the pipettes, wax paper or plastic. Save the
glass beaker, the pipette and pump, your basket and anything else that is reusable.
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QUESTIONS: Use information from your notes and this lab to answer the following questions.
Do not copy the information directly from the packet, put it into your own words.
Type only if your handwriting is difficult to read. Black ink only.
1) What was the source of DNA in this lab? __________________________________________________
2) Approximately how much DNA is in the sample you extracted? (example: 1 strand, 46 strands, thousands
of strands, etc.) Explain your answer.
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3) What was the role of the salt in this experiment? Explain how it works.
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4) What was the role of the detergent in this experiment? Explain how it works.
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5) What was the purpose of pouring the cold alcohol over the cheek cell solution? Explain.
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6) Would the DNA appear different in the alcohol vial if we had used plant DNA instead of human DNA?
Explain by describing the difference between plant and animal DNA. Are those differences visible?
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7)
Rough handling or decomposition may cause random breaks in the DNA evidence that is collected from a
crime scene. For example, bacteria that feed on the body will produce enzymes that cause breaks in the
DNA. The PCR process cannot amplify a broken segment of DNA. If a target sequence is broken, you
cannot make a proper DNA Profile. You are going to model damage to DNA.
a) Draw 3 or 4 random breaks along the in the DNA below (one has been drawn in for you).
b) Now draw in two, gene segments. One is this long, ‘___’ the other is this long
‘___________________________________’. Draw them in anywhere you want as long as they
are not on top of each other. Do not concern yourself where they land compared to the breaks.
Break
Now that you have completed your model, consider this:
There are many different types of non-coding, repeat regions in human DNA. Some, called Variable Number
Tandem Repeats (VNTR’s) are as long as 3000-5000 base pairs. Short Tandem Repeats (STR’s) are much
shorter. They may only be somewhere between 50-500 base pairs long. Both VNTR’s and STR’s could be
used to differentiate between individuals.
Why do you think that STR’s are used by Forensic Scientists to create a DNA Profile instead of the longer,
VNTR’s. Explain.
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Thermocycler used to conduct PCR
The apparatus holds DNA samples in very small test tubes that fit under the
top lid of the cycler. It automatically heats and cools the samples. The
heating cycle breaks the strands of DNA so that primers can attach to the
strands outside the target sequence. The cycler then cools the DNA while
DNA polymerase enzyme attaches nucleotides to amplify the DNA. The
cycler times the heating and cooling cycles so that optimum amplification
of target sequences can take place.
Gel Electrophoresis is used to
separate target sequences of different
lengths so they can be visualized
Once the target sequences have been
amplified, they can be separated to
determine their length using gel
electrophoresis. A dye will help to
visualize bands of DNA.