Download A2.1.4.GeneticTesting

Activity 2.1.4: Genetic Testing (Optional)
Introduction
Aaron and Gina Smith decide to have genetic testing to determine if they are carriers
for cystic fibrosis. They both feel this is information they need to know before they
make decisions about having children. Results reveal that neither Gina nor Aaron is
a carrier for the disease. Relieved to know they cannot pass the cystic fibrosis gene
on to a child, the couple begins plans for their new family.
Multiple tests and interventions are available to test and screen our DNA. In this lab,
you will experience one method of looking inside of our cells and decoding the
message buried in the sequence of nucleotides. The genotype, what is written in our
DNA, predicts phenotype, what we see as a result of that code. This genotype may
relate to a simple trait, such as eye color, or presence of dimples, or, unfortunately,
may signal the presence of an incurable disease.
Amazingly, all humans share 99.9% of their genetic code. It is the remaining 0.01%
that actually makes us unique individuals. This small percentage controls aspects of
our appearance, our personality, and even our health profile. The parts of the human
genome that vary by just a single nucleotide are referred to as single nucleotide
polymorphisms (abbreviated SNPs and pronounced “snips”). If these SNPs occur in
a non-coding region of the genome, you would not even know they are there;
however, SNPs in an important gene can lead to variation in traits or even disease.
Remember that a change in DNA can lead to a change in a protein. If the protein
plays a role in keeping you healthy, serious consequences may occur. Tests to
locate these SNPs can pinpoint disease genes and provide medical options.
In the 1930s, scientists first studied a genetic basis to taste. While synthesizing a
chemical called phenylthiocarbamide (PTC) in his lab, scientist Arthur Fox
accidentally released some into the air. Fox’s lab mate complained that the dust had
a very bitter taste. Fox, however, tasted nothing. After further studies, scientists
concluded that the inability to taste PTC is actually a recessive trait. Bitter-tasting
compounds are recognized by receptor proteins on the surface of taste cells. The
gene for the PTC taste receptor, TAS2R38, was identified in 2003. Sequencing
identified three variations in this gene from person to person. These base pair
differences, or SNPs, correlate to a person’s ability to taste PTC. In this lab, you will
have a chance to examine DNA sequences, determine genotype, and use this
information to predict phenotype. This type of genome screening allows individuals
to pinpoint SNPs and to use this genetic information to prevent and treat disease, as
well as design new drugs tailored to your own genetic profile. But more on that later!
© 2010 Project Lead The Way, Inc.
MI Activity 2.1.4: Genetic Testing (Optional) – Page 1
Sue Smith and four of her college friends have volunteered to donate their DNA to
the molecular biology department for genetic testing. You will look into their DNA,
locate a region where a SNP may occur, and predict their ability to taste PTC. Using
knowledge of laboratory techniques you have learned in the Biomedical Sciences
courses, you will discuss how to replicate a specific sequence of DNA, identify SNPs
using restriction enzymes, and analyze genetic differences on a gel. Gel results will
provide information regarding genotype. Identify the phenotype of each individual
undergoing testing and illustrate the connection between genes and their
expression.
Equipment



Computer with Internet access
Laboratory journal
Edvotek’s Genetically Inherited Disease Detection Kit #116
o Ready-to-load DNA samples
o Agarose powder
o Electrophoresis buffer
o Transfer pipets
o Practice gel loading solution
o InstaStain® Methylene Blue







PTC tasting paper and control paper
Highlighter
Gel staining equipment and reagents
Micropipettors and tips
Microcentrifuge tube rack
Electrophoresis chamber and power supply
UV transilluminator or white light box
Procedure
Part I: Using SNPs to Identify a Gene of Interest
Before scientists can amplify the gene responsible for PTC tasting ability, they need a
sample of each student’s DNA. Sue and her friends Lena, Jose, Rebecca, and Winston
head into the lab to donate a DNA sample from their cheek cells.
1. With a partner, brainstorm how you can isolate DNA from your own cheek cells.
Thinking about where DNA is located in the cell, describe what has to happen for
you to isolate cells and release the DNA from these cells. Write down your ideas
in your lab notebook and discuss your ideas with the class.
2. Research the process of DNA isolation using reputable sources on the Internet.
Identify a simple procedure for extracting DNA from human cells. List at least
three chemicals or reagents used in the process and describe how each helps
release pure DNA.
Now that the lab has the necessary DNA, PCR is used to target and amplify the gene
you will be working with in the lab. The gene of interest in the experiment, TAS2R38, is
located on chromosome #7. This gene is associated with our ability to taste the
© 2010 Project Lead The Way, Inc.
MI Activity 2.1.4: Genetic Testing (Optional) – Page 2
chemical PTC. Primers specific to a 220-bp sequence in this gene were used to make
millions of copies for our study.
3. Observe the specific PCR protocol shown below. Referring to this specific
laboratory experiment, describe what is happening in the tube at each
temperature in your laboratory journal.
o Denature at 94°C for 30 seconds
o Anneal at 68°C for 30 seconds
o Extend at 72°C for 30 seconds
Restriction enzymes, molecular scissors, recognize specific DNA sequences and cut
the nucleotide strands. In this experiment, a specific restriction enzyme, HaeIII, has
been used to identify SNPs or base pair differences in the amplified segment of the PTC
tasting gene. Since DNA codes for the production of proteins, changes in the sequence
can lead to differences in the protein – in this case, the receptor that attaches to the
chemical and allows a person to taste PTC.
4. Observe the DNA sequences of a nontaster, a person who can not taste PTC,
and a taster, a person who can taste PTC, shown below. NOTE: This is only a
region of the 220bp PCR product – a region that contains a SNP.
NONTASTER (tt)
TASTER (TT)
GGCGGGCACT
GGCGGCCACT
CCGCCCGTGA
CCGCCGGTGA
5. Copy the sequences into your laboratory journal. Note any differences using a
highlighter.
6. In your laboratory journal, show how the two sequences of amplified DNA would
be affected by digestion with the enzyme HaeIII. This enzyme recognizes the
DNA sequence
7. Note that the line above shows exactly where the restriction enzyme makes its
cut. Notice that HaeIII cuts DNA evenly down the strand and leaves even edges,
referred to as blunt ends. Other enzymes, such as EcoRI, one of the enzymes
you used in the DNA Detectives lab in HBS and in your paper plasmid
experiment in Unit 1, cut DNA unevenly, leaving jagged edges called sticky ends.
8. In your laboratory journal, describe how the enzyme would cut in a taster and in a
nontaster. How many fragments would be produced in each case? Draw or
explain how this product would be verified using gel electrophoresis.
9. Predict what you would see on a gel if a person is a heterozygous Tt taster.
10. Discuss your predictions with the class.
Part II: Analyzing PCR Products by Gel Electrophoresis
© 2010 Project Lead The Way, Inc.
MI Activity 2.1.4: Genetic Testing (Optional) – Page 3
The PCR samples from each student have been digested with the restriction enzyme
HaeIII. You will now run the digested products on a gel to determine genotype for the
ability to taste PTC.
11. Prepare the gel as directed by your teacher.
12. Place the solidified gel in the electrophoresis chamber and add enough 1X
electrophoresis buffer to cover the surface of the gel.
13. Carefully remove the comb. Make sure that the wells are submerged. If
necessary, add additional buffer.
14. Using a micropipettor and a fresh tip for each sample, load the following onto
your gel:
o
o
o
o
o
o
Lane 1 – Undigested PCR product – 35µl
Lane 2 – Sue’s sample – 35µl
Lane 3 – Lena’s sample – 35µl
Lane 4 – Jose’s sample – 35µl
Lane 5 – Rebecca’s sample – 35µl
Lane 6 – Winston’s sample – 35 µl
15. Copy your gel loading diagram into your laboratory journal.
16. Follow your teacher’s instructions to assemble the gel electrophoresis apparatus
and connect the power supply. Be sure to check the orientation of your gel. The
DNA containing wells should be closer to the negative pole and farther away
from the positive pole.
17. Run the gel at 100-130V for approximately 30 minutes or until the dye front has
moved almost to the end of the gel. Check your gel every ten minutes and turn
off the power supply when the dye is near the bottom of the gel.
18. Stain the gels according to your teacher’s instructions. This part may be
completed for you.
19. View the gel using a white light box or a UV transilluminator.
Part III Determining PTC Tasting Genotype and Phenotype.
20. Observe the bands on your gel or in a photograph of your gel.
21. Locate the lane containing the undigested DNA fragment. The undigested PCR
product corresponds with a size of about 221bp (the size of the fragment
amplified in the lab).
22. Compare the digested DNA of each student to the uncut control and determine
genotype. Refer to the predictions you made in Steps 8 and 9 about what you
would see on a gel if a person was a tt nontaster (homozygous recessive), a TT
taster (homozygous dominant), or a Tt taster (heterozygous).
23. Describe your findings in your laboratory journal. Predict whether or not each
person will be able to taste PTC.
24. Follow steps 26 - 30 to test your own ability to taste PTC.
25. Obtain one strip of control taste paper from your teacher.
© 2010 Project Lead The Way, Inc.
MI Activity 2.1.4: Genetic Testing (Optional) – Page 4
26. Place the control strip on your tongue for several seconds. Note the taste in your
laboratory journal.
27. Remove the control paper and dispose of it in the appropriate waste receptacle.
28. Obtain a PTC taste paper from your teacher and place it in the center of your
tongue for several seconds.
29. In your laboratory journal, describe the taste of the PTC paper. Compare the
taste to that of the control. Is the taste strongly bitter, weakly bitter, or the same
as the control paper?
30. Correlate your PTC phenotype to your possible genotypes. What would you see
on a gel if you were able to test your own DNA? Explain your reasoning in your
laboratory journal.
31. Draw a flow chart in your laboratory journal or use Inspiration software to
describe the process you would use to extract your own DNA and determine your
own PTC genotype. Pay attention to the role of SNPs in your analysis and
mention all relevant lab steps. Show how each step of the lab will help you make
your final conclusion about your own genotype.
32. Read the Genetic Science Learning Center article PTC: Genes and Bitter Taste
at http://learn.genetics.utah.edu/content/begin/traits/ptc/. Use this
information to help you interpret your results and answer the Conclusion
questions.
Conclusion
1. What is the role of PCR in this genetic testing experiment?
2. Explain how the HaeIII enzyme discriminates between the C-G polymorphism in
the TAS2R38 gene.
3. Using what you know about genetics, SNPs, and the PTC gene, explain why it is
possible for a person to be a “weak taster.”
© 2010 Project Lead The Way, Inc.
MI Activity 2.1.4: Genetic Testing (Optional) – Page 5
4. How can the techniques described in this lab be used to test for human disease
genes? Would this type of testing work on every disease with a genetic
component? Why or why not?
5. Another student at the college, Bob, returns to the lab for his PTC test results.
The gel shows that he is a homozygous recessive non-taster. However, when
Mick tastes the PTC paper, he obviously can taste the bitter chemical. Provide a
possible explanation for this difference.
6. What ethical issues are raised by human DNA typing experiments?
7. Some studies have shown that PTC “tasters” are less likely to become smokers.
Why do you think scientists are seeing this correlation?
© 2010 Project Lead The Way, Inc.
MI Activity 2.1.4: Genetic Testing (Optional) – Page 6