Name: ____Teacher
Biology
Key_____________
In The Year 2525…
Using Light-Cycler/Real Time PCR Data to Analyze a Founder Scenario.
Introduction:
Over the past thirty years we have witnessed a phenomenal growth in our ability to both
collect and assess genetic information. Biologists have determined the entire genetic sequence
for many individual species. Likewise, biologists are becoming increasingly adept at using this
information to evaluate the genome of specific individuals. Technology exists that can be used
to determine your specific genotype for a number of different traits.
The light cycler, or real time polymerase chain reaction (PCR), is one piece of equipment
that is used for such diagnostic purposes. Using PCR, fluorescent molecular tags, and the
knowledge of single nucleotide polymorphisms (SNPs) within an allele, the light cycler can
determine the homozygosity or heterozygosity of an individual for a specific allele. The device
relies upon the “melting” of DNA, or the temperature at which the two strands of DNA will
denature and separate from one another. During the light cycler/PCR process, fluorescent
markers attach to the DNA double helix at a SNP. The light cycler device records this
fluorescent glow. The temperature of the DNA sample is then continually raised until the two
strands denature. At this point the fluorescent markers let go of the DNA and stop “glowing”,
also measured by the light cycler. Different base pairs within DNA will “melt” at different
temperatures, depending upon the hydrogen bonds between them. Remember, also, that alleles
are different due to differences within the sequence of bases of which they are comprised.
Fluorescence
Fluorescence
Check: Based on the above information:
Predict what a graph of fluorescence data will look like for
A heterozygous individual.
A homozygous individual.
Temperature (C)
The general idea
is that the
heterozygote will
have two peaks
and the
homozygote only
one.
Temperature (C)
Given the title of the activity, you may be wondering why all the fuss over the light
cycler? Well, you know that Hardy-Weinberg can be used to calculate the frequency of alleles
within a population and to predict the expected percentage of heterozygous and homozygous
individuals. In the 70 or so years since Hardy-Weinberg has been widely applied to living
populations, data on allele frequency has been obtained through the analysis of phenotypes
through the context of a pedigree… much like what we have done in class. However, with
technology such as the light cycler, biologists can now screen the DNA itself for SNPs and
determine the genotype of an individual. This data, compiled for a population, can then be
analyzed using the Hardy-Weinberg model. You will do this in the following activity.
KEY
Biology
Review/ Pre-Lab: Before you begin the activity, answer the following questions.
1. Define the following terms:
a. Allele – One of two or more alternate forms of a gene.
b. Genotype - The genetic composition of a living organism, or the pair of alleles
that a diploid organism possesses for a particular trait.
c. Single nucleotide polymorphism (SNP) – A specific location within a gene for
which the specific base present may differ among individuals.
d. Hardy-Weinberg equilibrium – A state in which the allele frequencies within a
population remain constant. Assumes random mating, no natural selection, a
large population, no immigration or emigration, and no change in allele
frequency due to mutation.
e. Founder effect – A circumstance in which a population possess the unique genetic
traits and characteristics of an original and relatively smaller founding
population.
f. Genetic drift – A change in allele frequencies from one generation to the next,
based on the random chance involved in determining which alleles parents pass
on to offspring.
2. Describe a reason why genetic equilibrium may shift from one generation to the next.
Please be specific.
Answers here will vary, but students should discuss one reason with supporting details.
This might include the fact that mutations are known to occur at a predictable rate, the
reality of migration, the selective pressures exerted by the environment, the reality that
not all reproductive age members of a population will breed, and the role of chance and
crossing over during the production of gametes in individuals of reproductive age.
What this question really gets at is that populations will not remain at equilibrium and
that Hardy-Weinberg is then a way to track changes in allele frequency from one
generation to the next.
3. Describe three factors that have an effect upon genetic equilibrium.
Students may list any of the other factors listed in question 3.
KEY
Biology
Look at http://www.karymullis.com/pcr.shtml . This will describe the basic process of PCR, or
polymerase chain reaction.
4. In a few sentences, describe what PCR can do with a very small sample of DNA.
Essentially, students should see that the PCR process copies or amplifies a small sample of
DNA billions of times over in a relatively short period of time. This provides a volume of
DNA that can be analyzed in a lab setting.
5. Given your answer for question 4, why do you think PCR has drastically influenced the
field of forensic investigation (it has)?
Students should infer, from prior experiences, general cultural knowledge, and the PCR
animation, that even a miniscule sample of DNA from an investigation, crime scene, suspect,
etc. can be run through PCR. This then provides investigators with enough DNA to perform
analysis such as digestion with restriction enzymes and gel electrophoresis… the “DNA
fingerprint”
Look at the light-cycler handout which describes how a light cycler works.
6. For the light cycler/ real time PCR:
a. What is the advantage of using the light cycler (what can it do quickly and
cheaply)?
The light cycler can perform PCR quickly and at the same time determine the
presence of a specific SNP within a targeted gene. Essentially, it can determine the
genotype for a specific trait.
b. Why must other technologies be used alongside of the light cycler in genome
research (think of the limitations of the light cycler)?
You must know the genetic sequence of the target gene ahead of time. So, the light
cycler can only be used with known genes, it cannot discover new genes or unique
SNPs and nucleotide sequences.
Biology
KEY
Scenario: ...and now the fun part. 
In July of 1969, human beings first set foot upon the moon. In the years since, human
exploration of the solar system continues, primarily through robotic probes that send data back to
scientists on Earth. In fact, some scientists agree that continued robotic exploration may confirm
the presence of living organisms elsewhere in the solar system. This could even occur within our
lifetimes. Though robotic exploration is effective, there are those who feel that human
exploration should continue, with the planet Mars being the next target. Since 1969, engineers
have proposed a number of strategies for reaching the Red Planet. Generally, these include some
version of a 3 year time period spent in a vehicle the size of a mobile home, with a limited
amount of time spent poking around the surface of Mars. Yes, imagine the ultimate road
trip…except that leaving the car is never an option. Thanks goodness for iPods! 
Of the strategies for manned exploration of Mars, one of the more radical approaches has
been referred to as the “one-way-trip”. This is exactly what it sounds like; a person or select
group of people would volunteer to travel to Mars. Upon reaching Mars, they would stay there
permanently, spending the rest of their life exploring the planet and reporting back to Earth. This
person or group would be resupplied from time to time, but they would stay on Mars.
Biologically, a group of explorers embarking on a “one-way” mission could constitute a founder
population. This is, of course, if males and females of reproductive age were among the
explorers. If the “one-way” explorers were to have children, the eventual descendants of the
explorers would be similar in genotype to the founding explorers themselves. Your goal is to
analyze such a founding population and speculate as to how the genotypic makeup of the
descendants will compare to that of the initial founders.




Essential Questions:
Can you use the light cycler data to determine the genotype of each of the founding
Martian colonists (hypothetical colonists, of course)?
Can you calculate the frequency of alleles within the founding population?
Can you calculate the percentage of heterozygotes and homozygotes within this
population?
Can you use modeling software to predict the allele frequencies of future “Martian”
generations?
Task:
It is the year 2023 and the United Nations is organizing a “one-way” mission of
volunteers to study the planet Mars. Fifteen couples from around the world have signed up for
the mission. They will travel to Mars, begin study of the planet in depth, and set up a permanent
habitat on the planet. Your genetics study group has been contracted by the UN to assist in
developing a crew profile. You are responsible for generating data on relevant genetic traits of
the crewmembers. Likewise, the UN has asked you to write a summary outlining the genetic
makeup of future generations of children born on Mars. Remember that the original colonists
will be considered a founder population. Good luck!
KEY
Biology
Mars Project: Data Task One.
Genotypes.
Target Trait: Eye color, blue vs. non-blue
Target Gene: HERC2, SNP rs12913832
Task Summary: The Mars Project crew will travel to Mars as two separate groups.
Review the light cycler data for each contingent and determine their genotype for the
target trait. Record results in the table below.
Crew Contingent One
Crew Member
Molecular Genotype
(gg, GA, or AA)
Hetero or Homozygous
Phenotype, Blue or
Not-Blue
One
GA
HETEROZYGOUS
NOT-BLUE
Two
GA
HETEROZYGOUS
NOT-BLUE
Three
GA
HETEROZYGOUS
NOT-BLUE
Four
AA
HOMOZYGOUS DOM.
NOT-BLUE
Five
GG
HOMOZYGOUS REC.
BLUE
Six
AA
HOMOZYGOUS DOM.
NOT-BLUE
Seven
GG
HOMOZYGOUS REC.
BLUE
Eight
GG
HOMOZYGOUS REC.
BLUE
Nine
GA
HETEROZYGOUS
NOT-BLUE
Ten
GA
HETEROZYGOUS
NOT-BLUE
Eleven
GG
HOMOZYGOUS REC.
BLUE
Twelve
GA
HETEROZYGOUS
NOT-BLUE
Thirteen
GG
HOMOZYGOUS REC.
BLUE
Fourteen
AA
HOMOZYGOUS DOM.
NOT-BLUE
Fifteen
GA
HETEROZYGOUS
NOT-BLUE
Sixteen
GG
HOMOZYGOUS REC.
BLUE
Seventeen
GG
HOMOZYGOUS REC.
BLUE
Eighteen
GG
HOMOZYGOUS REC.
BLUE
Nineteen
GG
HOMOZYGOUS REC.
BLUE
Twenty
AA
HOMOZYGOUS DOM.
NOT-BLUE
Twenty One
GA
HETEROZYGOUS
NOT-BLUE
Twenty Two
GG
HOMOZYGOUS REC.
BLUE
Twenty Three
GG
HOMOZYGOUS REC.
BLUE
Twenty Four
GA
HETEROZYGOUS
NOT-BLUE
Twenty Five
AA
HOMOZYGOUS DOM.
NOT-BLUE
Twenty Six
GG
HOMOZYGOUS REC.
BLUE
Twenty Seven
GA
HETEROZYGOUS
NOT-BLUE
Twenty Eight
GG
HOMOZYGOUS REC.
BLUE
Twenty Nine
GG
HOMOZYGOUS REC.
BLUE
Thirty
GG
HOMOZYGOUS REC.
BLUE
Crew Contingent Two
Mars Project: Data Task Two.
Data Totals.
KEY
Biology
Task Summary: Use the table below to count and calculate data totals and percentages
where indicated.
Data Totals.
Total Mars population,
both contingents
30
Total Number of alleles,
Both blue and non-blue
60
Frequency of each allele
within the Crew
Population
Total number of Blue
Alleles
40
40/60=0.6667 OR 66.67%
Total number of Non-blue
alleles
20
20/60=0.3333 OR 33.33%
Mars Project: Data Task Three.
Hardy-Weinberg Analysis.
Task Summary. Given the frequency of each allele within the population of the Mars
Crew, calculate the expected percentage of homozygous dominant, heterozygous, and
homozygous recessive individuals. Remember that the Non-blue allele is dominant over
blue allele.
In the space below, calculate p2, 2pq, and q2.
Show work please.
ASSUMING P IS NON-BLUE AND Q IS BLUE
P2= 0.33332= 0.11 or 11%
Q2= 0.66672 = 0.44 or 44%
2pq = 2 x 0.3333 x 0.6667 = 0.44 or 44%
Next, use your data from part one to calculate the actual percentage of each genotype
within the thirty-person Mars crew. Please show work.
Heterozygous
= 10/30 = 0.33 or 33%
(expected is 2pq, 44%)
Homozygous not-blue = 5/30 = 0.167 or 16.7% (expected is p2, 11%)
Homozygous Blue = 15/30 = 0.50 or 50%
(expected is q2, 44%)
Finally, compare the predicted Hardy-Weinberg values to the actual values.
following in a few sentences.

Are they the same or are they different?
The values ate slightly different
Answer the
KEY
Biology

What might explain any similarity or difference between the predicted and
actual values? Hint, think about how the crew was chosen. Crew members were
analyzed for eye color but eye color was not a criteria for inclusion in the
final mission crew.
As this is intended for an intro level biology class, I would not ask students to
calculate Chi-square. However, performing a Chi-Square test suggests that the
difference is likely the result of random chance. The hint is meant to help
students infer that, in regard to eye color, the crew members are a random sample
of the population.
Mars Project: Data Task Four.
Population genetic analysis.
Task summary: In this step, you will use ‘Allele A1’ software to analyze possible
allele frequencies for the descendants of the Mars Crew. Assume that the couples of
the crew will have children. These children will in turn will grow and have their own
children. As generations pass, what will happen to the allele frequencies within the
Martian population?
Proceed to
http://faculty.washington.edu/herronjc/SoftwareFolder/AlleleA1.html
Download and open Allele A1 as directed.
Allele A1 screen shot.
Click this arrow to
expand Data window.
Allele A1
simulates two
alleles.
Designate the
Non-Blue
allele as A1
and the blue
allele as A2
Set the nonblue
frequency
here… and
here.
Set your starting
Click
set is
to a “closed
Set this
as this
0.02.arrow,
As this
population
here, this is
’50 generations’
population”
it is possible
the aCrew.
child
the Mars
bearing couple may end up sharing a common
ancestor.
KEY
Biology
Once you have set your parameters within Allele A1, you will simply click “Run” in
thew bottom left to run a simulation. This will demonstrate what occurs to the
frequency of the two alleles over a span of 50 generations.
Simulation One:

Run the simulator once and answer the following:
Describe what has happened to the frequency of the two alleles.
One of several possibilities may occur here. With most of the scenarios in
Allele A1 either one of the two alleles will become extinct within the Mars
population or their frequencies will fluctuate widely around an approximate
equilibrium point.

Did the population eventually reach an equilibrium for the blue/non-blue
eye trait?
It may, but with the size and parameters for the Mars population, most likely
only by one of the two alleles completely dying out within the Mars population.

Why may you have obtained the results you did for this simulation?
Remember the factors that effect Hardy-Weinberg equilibrium.
Answers may vary. Essentially, students should cite ways in which the Mars
population would not meet the conditions for Hardy-Weinberg Equilibrium. Also,
students should discuss that with such a small population the effect of genetic
drift in particular will be more pronounced. This is especially so if one of
the alleles died out.
Simulation Two:

Run the simulation again.
Describe what has happened to the frequency of the two alleles.
Look for answers similar to simulation one
KEY
Biology

Did you get the same or very similar results as the first simulation?
Why or why not?
Look for answers similar to simulation one. If results are different, students
should cite the random nature of genetic drift and the pronunced effect genetic
drift will have in such a small population from one generation to the next,
Simulation Three: Run the simulation a number of times until you get a result that is
drastically different than either of the first two simulations.

Describe what has happened to the frequency of the two alleles.
Students should describe results that are the oppositr of what they obtained in
the first two simulations. See simulation one for an explanation.

What may have happened with successive generations of the Mars colonists
to make these results differ from those of your first two simulations?
Please be as specific as you can.
Students should infer that events such
eliminate mating partners (perhaps the
natural selection in the harsh Martian
different in each scenario. With such
play out very differently.

as non-random mating, random events to
death of a crew member), mutation,
environment, etc.may have been slightly
a small population, each scenario can
What role does genetic drift play in each of the Mars Population
simulations and why is its effect so pronounced?
Genetic drift causes the seemingly drastic fluctuations in the frequency of
each allele and, in simulations where It occurs, the extinction of one of the
two alleles. Because the population is so small, there is little population to
“buffer” what would otherwise be small and insignificant changes.
Simulation Four: Remember that the Mars Project Crew is a relatively small population
of 30 people. Change the parameters of Allele A1 so that it reflects a typical
KEY
Biology
population. Use the same allele frequency, but set the population to at least
1,000,000 people. Run the simulation again.

What occurs in this simulation that does not occur in any of the Mars
Crew Member simulations?
The frequency of each allele is maintained at a relative equilibrium. The
initial frequency of the non-blue allele, for example, changes very little over
the 50 generations.

In terms of Hardy-Weinberg, how does this final simulation differ from
those involving the Mars Crew members?
The large size of the population allows for small changes to offset one
another, thus resulting in what appears to be a relative genetic equilibrium.
Final Summary: Summarize your findings to the United Nations below. Be sure to detail
the genetic make-up of the initial Mars Crew in terms of the target trait.
Also, describe how, according to models of population genetics such as HardyWeinberg, the Mars population will change over successive generations.
Answers here



will vary of course. However, I would look for the following:
A concise summary of the light-cycler genotype data with examples.
A concise summary of the Allele A1 simulations with examples
AN interpretation of the data and simulation results in light of the
Hardy-Weinberg principle and knowledge of population genetics.

Overall quality of writing. I encourage students to write a coherent
paragraph, not simply a series of sentences that sound as though they are
merely answering individual questions.
In terms of scoring this, I would adapt any existing writing rubric that you may
already use with your students.
______________________________________________________________________________________
__________________
______________________________________________________________________________________
__________________
______________________________________________________________________________________
__________________
______________________________________________________________________________________
__________________
______________________________________________________________________________________
__________________
______________________________________________________________________________________
__________________
KEY
Biology
______________________________________________________________________________________
__________________
______________________________________________________________________________________
__________________
______________________________________________________________________________________
__________________
______________________________________________________________________________________
__________________
______________________________________________________________________________________
__________________
______________________________________________________________________________________
__________________
______________________________________________________________________________________
__________________
______________________________________________________________________________________
__________________
______________________________________________________________________________________
__________________
______________________________________________________________________________________
__________________
______________________________________________________________________________________
__________________
Finally…bonus question. The title of this activity refers to an older and relatively
obscure song that was, for a brief time, a number one hit. For a few bonus points
research and tell me the following:

What is the song and who is the artist?

When was it on top of the music charts?

What other very significant event occurred when this song was a number one hit.
Yes, you must have all three for credit.

You may adapt or omit this question. Answers are as follows:
Song: “2525”
Artist: “Zager & Evans” It was a one hit wonder.
Time on music charts: Billboard Top 100, July and August 1969
Other Very Significant Event: The Apollo 11 Moon Landing occurred in July 1969.