PCR AMPLIFICATION OF CHEEK CELL DNA
LAB MBDNA3
From Chromosome 16: PV92 PCR Informatics Kit, Biotechnology Explorer, BioRad, 2004, and
Isolation and PCR Amplification of Human Chromosomal DNA, Lab 4a-4b, Science and Ethics of
the Human Genome Research, Spring 2005, Prof. Joshua Corrette-Bennett, Westminster College.
INTRODUCTION
DNA
DNA is found in all living organisms. It contains the genetic code for making proteins –
especially enzymes – that are necessary for the production of all other molecules in the
cell. Thus DNA is the “master molecule” of the cell. It is responsible for passing traits
from one generation to the next and allows species to adapt over time. While DNA is
fairly consistent in structure and function in all cells and organisms, variations in the
genetic code can and do exist. DNA exhibits variations known as polymorphisms. The
majority of these polymorphisms occur within the base-pair sequences and range from
single base-pair alterations (different base-pairs, deletions, insertions, etc.) to large
insertions or deletions of DNA sequence (thousands of base-pairs). These variations can
be analyzed using current biotechnology to reveal important information about an
individual or groups of individuals.
DNA Polymorphisms and the PV92 Locus
Every human genome contains short repetitive interspersed elements (also known as
SINEs). Each SINE consists of a relatively short sequence, anywhere from ten to a few
hundred base-pairs in length, and exists at numerous places throughout the genome.
These repeated sequences have been randomly inserting themselves throughout our
genomes for millions of years. Over 44% of the human genome consists of repetitive
DNA sequences! One such SINE is the Alu sequence, a 300 base-pair stretch of DNA
sequence that exists at more than 500,000 places throughout the human genome! The
origin and function of this SINE is not completely understood, but it does not code for
any type of protein or RNA.
We will analyze the Alu SINE at one specific location, called the PV92 locus, on
Chromosome 16. Not everyone has the Alu repeat at this position of chromosome 16.
This particular genetic locus is dimorphic (two forms). That is, the Alu element is either
present or absent at this particular position on each chromosome 16. Some people may
have the Alu repeat inserted at the PV92 locus on both chromosomes (homozygous +/+),
some may not have the Alu repeat in either chromosome (homozygous -/-), and some
may have the Alu repeat in one, but not the other (heterozygous). The presence or
absence of the insert can be detected using the PCR reaction followed by agarose gel
electrophoresis.
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Figure 1. The presence or absence of the Alu insert within the PV92 locus on Chromosome 16.
PCR Amplification of DNA
During the mid-1980’s, a creative scientist developed an elegant and powerful technique
to generate millions of copies of a specific DNA sequence. This technique is called
polymerase chain reaction amplification, or PCR amplification. It amplifies, or copies, a
specified region of DNA sequence. This is particularly useful when there is very little
sample to begin with, there has been some degradation of template, or when the amount
and complexity of DNA sequence being searched is very high. PCR amplification
produces many copies of target DNA in quantities sufficient for detection and analysis by
agarose gel electrophoresis. PCR is most efficient at amplifying fragments of DNA that
are a few thousand base pairs (bp) or less, but technology in this field is constantly
improving and can now provide amplification of much longer pieces (up to 21,000 bp).
PCR is analogous to photocopying: we start with an original (the desired DNA template
or target sequence), use the appropriate machinery (a thermal cycler) and reagents (DNA
polymerase, nucleotides, primers), and produce millions of identical DNA sequences, just
as a Xerox machine can produce many copies of an original document. Reactions that
occur in a PCR tube are equivalent to what occurs in the nucleus of a cell every time the
cell replicates, or synthesizes, chromosomes in preparation for cell division. Many of the
same components used by the cell are included in the PCR reaction, but the PCR tube
contains only the bare minimum of components necessary for DNA synthesis.
Primers are a critical component of DNA replication and thus PCR, providing specificity
for copying the desired region. A primer is a short (12-50 base-pair), single-stranded
sequence of DNA capable of pairing with DNA sequences on either side of the desired
target sequence. Often primers are referred to as oligonucleotides. Two different primers
are used for PCR. The primers should bracket the target region and direct the synthesis
of new DNA strands in the direction of the other primer (towards each other). The
primers provided for this PCR reaction are specific for DNA sequences that flank either
side of the PV92 site on human Chromosome 16, regardless of whether the Alu insert is
present or not (Fig. 2). If the Alu insert is present at the PV92 locus, the PCR reaction
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will amplify a piece of DNA that is 941 bp long. The absence of the Alu insert produces
a 641 bp DNA fragment.
Alu
After PCR:
Alu
Alu
Alu
941 bp
641 bp
Fig 2. The PV92 locus on Chromosome 16. The arrows represent the primers used in the PCR
reaction. Two different size DNA fragments are produced with these primers, depending on
whether the Alu insert is present (941 bp) or absent (641 bp).
Once the primers have bound to their complementary sequences, DNA polymerase uses
each primer as a starting point for synthesis of a new DNA strand. In this way, the two
strands of the double-stranded target region are bracketed by the two primers and
replicated simultaneously.
Template DNA must be included in the reaction. It can be obtained from many human
tissues such as cheek cells, a hair follicle, skin cells, or blood. In theory, just a few cells
or bits of tissues will supply enough DNA for the reaction but PCR requires more than
just one or two strands of DNA template. While PCR is capable of amplifying crude
DNA preparations and samples that have experienced some degree of degradation, DNA
samples that have experienced significant levels of DNA degradation (e.g., samples that
are millions of years old) do not make good templates.
All four types of nucleotides (called nucleoside triphosphates; deoxyadenine [dA],
deoxycytosine [dC], deoxyguanine [dG], and deoxythymine [dT]) are required for the
reaction. These are the building blocks that will be used by DNA polymerase to
construct the new DNA strands.
Buffer is required to provide and maintain the correct environment for the DNA
polymerase enzyme to function. Varying the concentration of the magnesium can also
affect the efficiency of PCR, but we will be keeping the concentration constant.
Taq DNA polymerase is the enzyme which performs the DNA synthesis. This is a
special polymerase, isolated from a thermophilic bacterium (inhabits volcanic hot springs
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or deep sea thermal vents) bacteria. At high temperatures (70°C - 95°C), this type of
DNA polymerase does not denature as rapidly as other enzymes so it does not lose its
activity as rapidly. Try to imagine how well your body would function in a 70°C or 90°C
water bath! As with any DNA polymerase, this enzyme joins individual nucleotides to
the growing DNA strand, synthesizing DNA in one direction. The enzyme can survive
multiple rounds of high temperatures used for denaturing double-stranded DNA. Not
only can the enzyme survive higher temperatures, its peak activity occurs at higher
temperatures (72°C).
Temperature cycling is essential to PCR. After the template DNA and all reagents have
been mixed, the cycling process of PCR begins. There are three different temperature
steps to each cycle of PCR.
One PCR Cycle
Denature DNA
Anneal Primers
Synthesize New DNA
94ºC
60ºC
72ºC
The first step of the cycle requires very high temperature in order to completely denature
the two strands of the template DNA. In the second step, the temperature is lowered to
allow the primers to anneal to the template DNA. Primers that remain bound to their
complementary DNA target sequence at higher temperatures are preferred because high
annealing temperatures allow for greater specificity, referred to as stringency. The
optimal annealing temperature for the primers provided with this kit is 60ºC. The primers
annealed to the template form a short double-stranded region which directs the Taq
polymerase to start synthesis at this site, thus targeting the amplified region. The final
step of the PCR cycle is performed at 72ºC, the optimal temperature for DNA synthesis
for the Taq polymerase. The PCR cycle shown above is programmed into the PCR
Thermal Cycler and is repeated 40 times during the experiment.
OBJECTIVE
Each student will isolate DNA from cheek cells and use it as template DNA in a PCR
reaction. The amplified DNA will be isolated on an agarose gel and analyzed. Each
student will be able to determine if he/she is homozygous (+/+), homozygous (-/-) or
heterozygous (+/-) for the Alu repeat.
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MATERIALS
Day 1
1.5 mL snap-cap microcentrifuge tube (micro test tube)
one screw cap micro test tube containing 200µL Instagene matrix
10 mL of 0.9% NaCl (saline) solution
200-1000 µL micropipettor & pipet tips
2-20 µL micropipettor & pipet tips
sterile cotton swab sticks
microcentrifuge
waste beaker
Kimwipes
Forceps
2 water baths (56ºC & 100ºC)
foam microcentrifuge tube holder
refrigerator
Day 2
0.2 mL microcentrifuge tubes containing 20 µL “Master Mix”
ice and ice buckets
2-20 µL micropipettor & pipet tips
PCR Thermal cycler
Day 3
Electrophoresis chamber
1% TAE agarose gel
1x TAE electrophoresis buffer
2-20 µL micropipettor & pipet tips
MMR (DNA size standards)
(+/+) PV92 locus control
(+/-) PV92 locus control
(-/-) PV92 locus control
individual PCR sample
sample loading dye
FastBlast agarose gel stain (1X)
Gloves
Staining trays
Labeling tape and Sharpies
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PROCEDURES
Day 1
DNA Isolation of Cheek Cells
►Under real research conditions, gloves would be worn at all times to prevent
contamination of samples. We will not be adhering to this rule, as it makes the
microcentrifuge tubes difficult to handle.◄
1. Label with your initials:
• one 1.5 mL snap-cap microcentrifuge tube (micro test tube)
• one screw cap micro test tube containing 200µL Instagene matrix
2. There should be small cups containing 10 mL of 0.9% NaCl (saline) solution at each
station. Select one of the cups and pour the yummy saline solution into your mouth, rinse
vigorously for 45 seconds, then expel (yes, spit) the saline solution back into your cup.
Food chunks will not make your experiment go well. If you have just eaten, rinse your
mouth with water before starting.
3. Take the 200-1000 µL micropipettor, set to 1,000 µL (1 mL), place a fresh microtip
onto the end, then transfer 1 mL of your saline solution/epithelial cheek cells into your
labeled snap-cap micro test tube. Make sure each person uses a fresh microtip for the
transfer process. Think about why this is important!
4. Take one of the sterile cotton swabs, dip the end you are not touching into the
remaining expelled saline solution in your cup and use it to swab the inside of one cheek.
Twirl the cotton swab vigorously while swabbing the cheek and gum area (don’t be
timid!). Then dip the swab into the snap-cap tube with the 1 mL saline/cheek cell
solution, twirling to dislodge any cells. Press the cotton swab up against the wall of the
tube while twirling to squeeze out as many cells and as much liquid as possible. Repeat
with a new cotton swab and the other cheek.
5. Place your micro test tube into one of the microcentrifuges. Be sure that your initials
are clearly labeled. Spin the tubes for 2 min. Do not begin the spin until all the slots in
the rotor have been filled! If all the slots are not filled, have your instructor check to be
sure the rotor is balanced.
6. Once the centrifuge has completely stopped, remove your tubes. A match-head sized
pellet of whitish cells should be visible. If you can’t see a pellet, carefully pour off
(decant) the saline solution into a waste beaker without disturbing the pellet. Refill the
micro test tube with another 1 mL of the saline/cheek cell solution and repeat the spin.
7. If a pellet is clearly visible, decant the saline solution into a waste beaker, being
careful not to lose your pellet. Blot the tube briefly on a Kimwipe. It is OK to have a
small amount of saline remain in the bottom of the tube, but it shouldn’t be more than 50
µL.
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8. Snap the top of the tube shut and loosen the pellet by flicking the tube with your finger
until clumps are no longer visible.
9. Using a 2-20 µL micropipettor set to 20 µL (plus a yellow microtip), transfer all of
your resuspended cells to your labeled screwcap micro test tube containing the Instagene
matrix. This may require repeated transfers. If the micropipettor is not sucking up your
cells, then they are probably not resuspended and you should flick the tube a few more
times. Make sure each person uses a fresh microtip for the transfer process.
10. Once you have transferred all of your cells, screw the cap tightly onto the tube and
then flick the tube again to thoroughly mix the contents.
11. Place the tube in a foam micro test tube holder (make sure your initials are clearly
labeled!) and incubate in a 56ºC water bath for 10 min. At the halfway time point (5
min), briefly remix the contents of the tube and quickly place the tube back in the water
bath for the remaining 5 min.
12. After the 10 min at 56ºC, briefly remix the contents by tapping the side of the tube,
put it back in the foam micro tube holder and place it immediately into the 100ºC water
bath. Be careful not to touch the water with your hands! You will burn yourself!
Incubate the tubes at 100ºC for 5 min.
13. Remove your tube from the 100ºC water bath and briefly mix the contents of the
tube. Pellet the matrix/cell lysate by spinning the tube in the microcentrifuge for 10 min.
As in Step 5, be sure the rotor is full or have your instructor be sure the rotor is balanced!
14. Remove your tube from the microcentrifuge and carefully store your sample in the
refrigerator overnight.
Day 2
PCR Amplification of the Target Region PV92
1. If your matrix/cell lysate sample has been refrigerated overnight, re-pellet the
contents by centrifuging the tube for 5 min.
2. Your instructor will provide very small (0.2 mL) micro test tubes containing the
correct amount (20 µL) of the “Master Mix”. The “Master Mix” contains the
nucleotides, buffer and Taq DNA polymerase necessary for the PCR reaction. The thin
walls of these tubes improve the efficiency of heat transfer to your sample during the
PCR amplification procedure. But this also means the tubes are fairly delicate and
pliable. Don’t squeeze them too hard! Each of you should take one of these tubes, label
it clearly with your initials and keep it in the ice bath provided!
3. Using a 2-20 µL micropipettor, transfer 20 µL of the cell lysate from the Instagene
screw cap tube into the PCR tube containing the Master Mix. ► When removing the
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lysate from the screw cap tube, be very careful NOT to transfer any of the Instagene
matrix into the PCR tube. Remove the lysate from the TOP of the liquid layer in the
Instagene tube. Your PCR reaction will not work if there is any matrix present.◄ Be
sure to dispense the lysate into the very bottom of the 0.2 mL tube containing the Master
Mix – this helps combine the two solutions.
4. Cap your tube and store it on ice until the instructor is ready to have you place your
samples into the thermal cycler. Place your sample tube in a capless tube adapter and
briefly centrifuge your sample (15 sec) so that everything is at the bottom of the tube.
5. When the instructor is ready, place all of the tubes from your group together in one
row of the thermal cycler block. Be sure they are clearly labeled! (You might want to
write down which row and what order the samples went in.) You are now ready to
amplify! The reactions will run for 40 cycles or roughly 3 hours. Your instructor will
provide you with the specifics of the PCR program.
The PCR samples will be removed from the thermal cycler and stored at -20ºC until the
class is ready to perform the agarose gel electrophoresis and analysis of the PCR
products.
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Day 3
Agarose Gel Electrophoresis
The instructor will hand out three PCR “positive control” samples to each group. One
positive control represents amplified products from an individual who is homozygous for
Alu inserts at the PV92 locus (+/+). The second control represents amplified products
from an individual who is homozygous for no Alu inserts (-/-). And the last positive
control represents amplified products from an individual who is heterozygous; one
chromosome contains an Alu insert while the homologous chromosome does not (+/-).
You will also be given one tube that contains loading dye (PV92 XC) and another that
contains your DNA size standards (“MMR”). It is crucial that you keep track of these
tubes and know what is required for each step.
1. Obtain your PCR amplified sample from the instructor. Place it in a capless tube
adapter and spin in the microcentrifuge for a few seconds. If all the spaces in the
centrifuge rotor are not filled, have your instructor check to be sure that the tubes are
balanced.
2. Add 10 µL of PV92 XC loading dye to your PCR tube and mix well by flicking the
tube. This dye serves a dual purpose, but it does not stain DNA. First, it increases the
density of the sample so that the entire sample will sink into the agarose gel well and
remain there. Second, it contains a dye that will migrate through the gel. The dye allows
you to visualize the progress of electrophoretic separation.
3. You will be given a pre-cast 1% agarose gel still in the plastic casting tray. Carefully
remove the tape from the ends of the casting tray and place the gel, still in the casting tray,
into the electrophoresis chamber. Pour enough of the 1X TAE buffer into the
electrophoresis chamber so that the gel is completely submerged. Remove the comb
slowly by pulling straight up on it. You must do this carefully so that the bottoms of the
wells do not rip. Orient the gel so that the wells are near the negative (black) terminal.
4. Your instructor will demonstrate how to load the agarose gel wells by loading the
Molecular Mass Ruler (MMR) sample for you. There are several things you should
remember when dispensing your samples into the gel well:
a. Do not stab the pipet tip into the bottom of the well. This will poke a hole in the
well and your sample will run out of the well and be lost in the buffer.
b. Be sure that the tip is placed into the very top of the well, just below buffer level.
If necessary, steady your hand on something (the lab bench, your other hand, etc.)
so that your sample will fall into the well. Do NOT lean the pipet tip against the
side of the well while dispensing the sample. I have watched several lab groups
rip the wells apart doing this.
c. When dispensing the sample, press down on the pipettor plunger slowly and
consistently. If you push the sample out too quickly, it will rebound out of the well
and be lost in the buffer.
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d. After loading the sample into a well, remember to keep the pipettor plunger
depressed until after your remove your tip slowly from the buffer. If you do not,
you risk sucking your carefully loaded sample back into the pipet tip!
e. Do not forget to record the order in which you load your samples! There is a
graphic of an agarose gel in the data section of the lab to do this.
5. Fill the pipet with the first control sample. Place the tip over the top of one of the wells.
The tip should be submerged in the buffer at this point. Holding the pipet steady, gently
dispense the sample into the well. The glycerin in the sample will allow the sample to sink
into the well. Do not place the pipet tip directly into the well or you will risk poking a hole
in the side or bottom of the well, and your sample may leak out of the gel. A new pipet tip
should be used for each sample. Each student should have a turn loading a well.
6. Load the following amounts of each sample into adjacent wells. Wait for the
instructor to give you gel loading instructions and make sure you change tips
between samples.
Lane
1
2
3
4
5
6
7
8
Sample
MMR (DNA size standards)
Homozygous (+/+) PCR control
Homozygous (-/-) PCR control
Homozygous (+/-) PCR control
Student 1
Student 2
Student 3
Student 4
Volume loaded
10 µL (whole sample)
10 µL (whole sample)
10 µL (whole sample)
10 µL (whole sample)
15 µL
15 µL
15 µL
15 µL
NOTE: Any group with five students should leave out the homozygous (-/-) PCR control
so that they have enough room for all five student samples in the gel. The (+/+) and
(+/-) PCR controls should provide you with enough information.
7. Secure the lid on the box. The lid will attach to the base and sit evenly in only one
orientation: red to red and black to black. Connect the electrical leads to the power
supply in the same manner: red to red and black to black.
8. Use of Power Supplies
a. Small power supplies: Attach the black power cord to the back of the power supply
and plug the supply into an electrical outlet. On the front of the supply, select “100V”
with the small black switch. Connect the electrophoresis chamber to the front of the
power supply, being sure to match the color of the leads, red-to-red and black-to-black.
Switch the power supply on using the black rocker switch. If the current is running
properly, there will be a curtain of bubbles rising from the negative (black) electrode at
the bottom of the electrophoresis chamber.
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b. Large power supplies: Before connecting the power supply to an outlet, make sure
the black rocker switch on the unit is set to “OFF”. Make sure the unit is set for voltage
(red light next to “V”) and then use the arrow keys to raise the digital read out from 0 to
100. Press the running figure on the far right hand side of the front panel and the digital
read out should quickly increase from 0 to 100 volts. To verify that is current running
through your electrophoresis chamber, look for a curtain of bubbles rising from the
negative electrode at the bottom of the chamber.
9. Run the unit for 40 minutes. After 5 minutes check your gel to make sure the
samples are running through the gel in the right direction (toward the red electrode)!
Remember, the dye that you can visualize is not stained DNA. It is used as a marker to
follow the progress of the samples through the gel.
Staining the Gel
1. After 40 minutes have passed, turn the voltage off and turn the black rocker switch off
at the back of the power supply. Then disconnect the electrodes from the power supply
and take the lid off of the electrophoresis chamber. CAREFULLY remove the gel and
casting tray making sure the gel does not slide out of the casting tray (cover both ends
with fingers because the gel can be very slippery and slide right out of the tray…splat!
There goes a week of work in the garbage).
It is recommended that you wear gloves for this phase of the lab, as the DNA dye will
stain your fingers blue.
2. Mark your tray with tape that has your initials on it. Do not write on the tray because
other lab sections need to use it.
3. For this type of staining, two groups will share one staining tray. Place the two
agarose gels in the staining tray by gently sliding the gel off of the casting tray.
4. Locate the bottle containing 1X DNA Fast Blast Dye solution and pour just enough
into the staining tray to cover the gels (~120 mL/staining tray).
5. Cover the gels with tinfoil, and let them stain overnight. If possible, store the gels in a
refrigerator during the staining, as it keeps the DNA bands clear and sharp. No
destaining will be necessary with this dye.
6. Gently pour the used DNA Fast Blast stain from the staining tray into a waste beaker.
Be sure the gels do not slip out! Carefully remove your gel and place it on some plastic
wrap. Place the gel on a light background (like white paper, light box or overhead
projector).
7. Determine whether you are homozygous (+/+ or -/-) or heterozygous (+/-) for the Alu
insertion. If the results are ambiguous, have your instructor help you interpret the gel
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DATA SHEET
Name ________________________
Period _______ Class ___________
Date ___________
Results of PCR Product Electrophoresis
Figure 1. Graphic Representation of PCR Product Electrophoresis
Draw a picture of what the stained agarose gel looks like. Use a ruler to mark the
distance (in mm) the samples have traveled from the bottom of each well.
Record order of
samples loaded here
Wells
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Table 1: Individual Sample Data from Agarose Gel
Record the numerical data obtained in Fig. 1 in the following table.
Lane
Number of
Bands
M
4 (maybe 5)
1000, 700, 500,
200, (100)
+/+
1
941
+/-
2
941, 641
-/-
1
641
Distance (mm)
Size (bp)
Student 1
Student 2
Student 3
Student 4
Table 2: Class Genotypes
Homozygous (+/+)
Heterozygous (+/-)
Homozygous (-/-)
# of students
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QUESTIONS & ANALYSIS OF RESULTS
1. What is the biological molecule that needs to be extracted from the cell for the PCR
reaction? What membrane(s) need to be broken to retrieve this molecule?
2. Define the term polymorphism. What type of polymorphism is a SINE?
3. You specifically looked at the Alu SINE located at the PV92 locus on Chromosome
16 in this experiment. What does is mean to say the SINE is dimorphic at this locus? If a
person is heterozygous for the Alu SINE, what would you find on each chromosome at
this locus (drawing a picture might help with this)?
4. Why are there nucleotides (dC, dA, dT, dG) in the master mix? What are the other
components of the PCR reaction?
5. From which organism was Taq DNA polymerase isolated? What is unique to this
enzyme that allows it to function in the PCR reaction?
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6. Describe the three main steps of each cycle of PCR amplification and what reactions
occur at each temperature.
7. What controls are run in this experiment? Why are they important?
8. Use Data Table 2 and the equations given below to determine the frequency of each
allele in your class sample Fill in the table below with your class data. Remember, a class
of 32 students (N) will have a total of 64 (2N) instances of each locus.
Hardy-Weinberg equation: p2 + 2pq + q2 = 1, therefore:
p = frequency of (+) allele = number of (+) alleles
total number of alleles (both + and –)
= 2(# of +/+ students) + (# of +/– students)
total number of alleles (both + and –)
= frequency of (+/+) students + ½ (frequency of (+/–)
students
q = frequency of (–) allele = number of (–) alleles
total number of alleles (both + and –)
= 2 (# of –/– students) + (# of +/– students)
total number of alleles (both + and –)
= frequency of (–/–) students + ½ (frequency of (+/–)
students
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Table of Class Allelic Frequencies
Category
Number
Frequency
(+) alleles
p=
(-) alleles
q=
Total alleles =
= 1.00
9. The following table contains data from a USA-wide random population study of
genotype at the PV92 locus.
Table of USA-wide Genotype at PV92 Locus
Category
Number
2,422
Homozygous (+/+)
5,528
Heterozygous (+/-)
2,050
Homozygous (-/-)
Total alleles = 10,000
Calculate the allelic frequencies for the US data as you did for the class data and fill in
the table below.
Category
Number
Frequency
(+) alleles
p=
(-) alleles
q=
Total alleles = 10, 000
= 1.00
How do the class allelic frequencies and the random US-wide allelic data compare?
Would you expect them to match? What reasons can you think of to explain the
differences or similarities?
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