Prepared by: Dr. K. L.

Hinkle
Associate Professor of Biology
Norwich University

Governors Institute of Vermont Winter

Weekend 2016
Biomedical Research Strand
Exploring Your Own Genome
This weekend you will get in touch with your
own genes by isolating your own genomic DNA!
Using cutting edge techniques regularly used
in biomedical laboratories, we will explore
what genetically makes us unique as
individuals (even though humans DNA is
99.9%). These tiny but significant differences
in our DNA sequence help determine our
individual traits and, ultimately, our distinct
identity. These little differences can determine
how susceptible we are to diseases, how we
respond to medications, and which traits we
carry. This weekend, we will examine one of the differences that
exist among individuals in DNA sequence that determines bitter
taste.
Timeline of events/experiments for the weekend (strand time):
Friday night (7-9pm): Overview of single nucleotide polymorphisms and
haplotype and why this is relevant to biomedical science and human disease.
Overview of experiments.
Saturday morning (9am-12pm): Isolate genomic DNA from saliva; Perform
polymerase chain reaction (PCR) of the bitter taste gene, TAS2R38
Saturday afternoon (3-5:30pm): Perform HaeIII restriction digest of PCR
products
Sunday (9am-12pm): Run PCR products through gel electrophoresis to
determine bitter taste phenotype genotype (and thus phenotype!); Discuss
results in the bigger context; come together as a group to determine how to
present results and significant findings at the closing ceremony
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Prepared by: Dr. K. L. Hinkle

Associate Professor of Biology
Norwich University

INTRODUCTION
Humans differ in their DNA sequences from one another by more than you
might think. Even though human DNA between individuals is 99.9% identical,
this still leaves more than 0.1% of the genome that differs. Turns out, there
are about 10,000,000 nucleotide differences between one individual
and the next! These single base differences are called single nucleotide
polymorphisms and account for genetic variability between individuals.
# base pairs (As, Ts, Cs, and Gs) in human genome:
~3,200,000,000 (3.2 billion)
# base pairs different between individuals: ~10,000,000 (10
million)

Sometimes, these SNPs (snips as they are called), do not lead to any
phenotypic difference, meaning that no noticeable change results in the
individual. However, sometimes the SNPs can be within gene coding regions,
causing alterations in traits in the individual. Examples of this are how some
people will react differently to a medication (e.g. Prozac) than others given
that they have differences in genotype.
These SNP differences between one individual and another are collectively
called haplotype. There are currently efforts to map these haplotypes
(HapMap) to understand SNPs that are related to diseases such as cancer,
Alzheimers, diabetes etc.; these efforts are also attempting to better deliver
personalized medicine, in that one person (due to their SNP variances)
may respond differently to medications (e.g. chemotherapy) than another
based on haplotype.
One routinely studied SNP that exists among individuals is the bitter taste
phenotype. There are several genes that are involved in bitter taste
sensation, but one gene, TAS2R28, is known to be important for bitter taste
sensation. There are three SNPs within the TAS2R28 gene that, when
individuals have one base or another, will elicit different bitter taste
sensation.
Mammals are believed to distinguish only five basic tastes: sweet, sour,
bitter, salty, and umami (the taste of monosodium glutamate). Taste
recognition is mediated by specialized taste cells that communicate with
several brain regions through direct connections to sensory neurons. Taste
perception is a two-step process. First, a taste molecule binds to a specific
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Prepared by: Dr. K. L. Hinkle

Associate Professor of Biology
Norwich University

receptor on the surface of a taste cell. Then, the taste cell generates a
nervous impulse, which is interpreted by the brain. For example, stimulation
of sweet cells generates a perception of sweetness in the brain. Recent
research has shown that taste sensation ultimately is determined by the
wiring of a taste cell to the cortex, rather than the type of molecule bound by
a receptor. So, for example, if a bitter taste receptor is expressed on the
surface of a sweet cell, a bitter molecule is perceived as tasting sweet.
Bitter-tasting compounds are recognized by receptor proteins on the surface
of taste cells. There are approximately 30 genes for different bitter taste
receptors in mammals. The gene for the PTC taste receptor, TAS2R38, was
identified in 2003. It was discovered that, while the overall sequence of
TAS2R38 is nearly identical from one individual to the next, there are three
nucleotides within different parts of the sequence that can vary among
individuals. Each variable position is termed a single nucleotide
polymorphism (SNP). One specific combination of the three SNPs, termed
a haplotype, correlates most strongly with tasting ability (e.g. if you have a
G there, youre a non-taster; if you have a C in that spot, youre a
taster). Analogous changes in other cell-surface molecules influence the
activity of many drugs. For example, SNPs in serotonin transporter and
receptor genes predict adverse responses to anti-depression drugs, including
PROZAC and Paxil.
In this experiment, a sample of human cells is obtained by saline
mouthwash. DNA is extracted by column isolation. Polymerase chain reaction
(PCR) is then used to amplify a short region of the TAS2R38 gene. The
amplified PCR product is digested with the restriction enzyme HaeIII, whose
recognition sequence includes one of the SNPs. One allele is cut by the
enzyme, and one is notproducing a restriction fragment length
polymorphism (RFLP) that can be separated on a 2% agarose gel. Youll first
test your genotype, predict your bitter tasting ability based on your
genotype, and then taste PTC paper to determine whether phenotype
matches genotype. Class results show how well PTC tasting actually
conforms to classical Mendelian inheritance, and illustrates the modern
concept of pharmacogeneticswhere a SNP genotype is used to predict drug
response.
Citations:

Prepared by: Dr. K. L. Hinkle

Associate Professor of Biology
Norwich University
Blakeslee, A.F. (1932). Genetics of Sensory Thresholds: Taste for Phenyl Thio Carbamide.
Proc. Natl. Acad. Sci. U.S.A. 18:120-130. Fox, A.L. (1932). The Relationship Between Chemical
Constitution and Taste. Proc. Natl. Acad. Sci. U.S.A. 18:115-120.
Kim, U., Jorgenson, E., Coon, H., Leppert, M., Risch, N., and Drayna, D. (2003). Positional
Cloning of the Human Quantitative Trait Locus Underlying Taste Sensitivity to
Phenylthiocarbamide. Science 299:1221-1225.
Mueller, K.L., Hoon, M.A., Erlenbach, I., Chandrashekar, J., Zuker, C.S., and Ryba, N.J.P.
(2005). The Receptors and Coding Logic for Bitter Taste. Nature 434:225-229.
Scott, K. (2004). The Sweet and the Bitter of Mammalian Taste. Current Opin. Neurobiol.
14:423-427.

Protocol #1: Isolating genomic DNA from saliva sample (from

QIAamp DNA Blood Mini Kit Procedures)
This procedure aims to isolate genomic DNA from your cheek/tongue
cells. Starting with a saline rinse, cells are isolated, washed, and
then taken through a lysis and DNA isolation procedure which will
ultimately yield genomic DNA that can be used in the next
experiment.
1. Collect cells by swishing with the 10ml of PBS provided for 1 minute;
spit the PBS back into the tube. Be careful not to spit mucous, just rinse
the mouth gently with the PBS.
2. Using a plastic transfer pipet, transfer 2ml of the saliva (without
bubbles) over to a 2ml microcentrifuge tube. Label the tube clearly on top
with your initials, date, and saliva.
3. Centrifuge samples at 1800 x g for 5 min.
4. Examine the pellet of cells at the bottom of the tube. Carefully decant
the supernatant by pouring it in the waste bucket.
___5. Repeat steps #2, #3, and #4, in the same tube, two more times. In this
way, we are collecting the appropriate number of cells from which to isolate
DNA.

Prepared by: Dr. K. L. Hinkle

Associate Professor of Biology
Norwich University

6. Using a P1000 pipette with the appropriate sized pipette tip, add
1000ul (1ml) of PBS to the pellet. Vortex the pellet well to resuspend.
7.
Centrifuge the samples at 1800 x g for 5 min.
7. Carefully decant the supernatant by pouring off the liquid into the
waste bucket.
8. Using a P200 pipette with the appropriate sized tip, add 180 l PBS to
your cell pellet. Vortex well to resuspend all of the cells.
10. Using either a P200 or P20 pipette, add 20 l QIAGEN proteinase K to
the sample. Proteinase K digests protein and assists in breaking down the
cells.
11. Using either a P1000 or P200, add 200 l Buffer AL to the sample.
AL buffer is a lysis buffer containing chaotropic salts that break down
cells.
12. Mix immediately by vortexing for 15 seconds.
13. Incubate in the water bath at 56C for 10 min. This is the optimal
temperature for the proteinase K.
14. Using either a P1000 or P200, add 200 l ethanol (100%) to the
sample, and mix again by vortexing. Ethanol precipitates DNA so that it will
come out of solution and will be able to attach to the column in the next
step.
15. Set your P1000 to 800ul. Carefully apply the entire mixture from
your 2ml tube to the QIAamp Spin Column without moistening the rim.
(There will not be 800ul total in your tube but we set the pipette at 800ul to
make sure to get all of it).
16. Close the cap of the QIAamp tube. Label your QIAamp Spin column
(top of the cap) with your initials. Centrifuge at 6000 x g for 1 min making
sure that all tubes are balanced in the centrifuge.
17. Place the QIAamp Spin Column in a clean 2 ml collection tube, and
discard the tube containing the filtrate in a waste bin.
18. Carefully open the QIAamp Spin Column and, using a P1000 pipette,
add 500 l of Buffer AW1. This is a wash buffer that will wash the high salt
concentration (from the lysis buffer) off the DNA.
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Prepared by: Dr. K. L. Hinkle

Associate Professor of Biology
Norwich University

19. Centrifuge at 6000 x g for 1 min.

20. Place the QIAamp Spin Column in a clean 2 ml collection tube
(provided), and discard the collection tube containing the filtrate in the waste
container.
21. Carefully open the QIAamp Spin Column and, with a P1000 pipette,
add 500 l of Buffer AW2. This is another wash buffer to further wash the
residual salts off the DNA on the column.
22. Centrifuge at full speed for 3 min. The full-speed spin removes all
traces of Buffer AW2 from the QIAamp Spin Column before elution.
23. Place the QIAamp Spin Column in a clean 1.5 ml microcentrifuge
tube (with a conical bottom) that has the cap cut off (provided), and discard
the collection tube containing the filtrate into the waste container.
24. Carefully open the QIAamp Spin Column. With a P100 or P200, add
100 l of water, being careful not to touch the pipet tip to the filter.
25. Incubate at room temperature for 1 min then centrifuge at 6000 x g
for 1 min.
26. Take the column out of the tube. Pipet up the 100ul that was eluted
and place it BACK on the column (this will increase the yield of DNA).
27. Incubate at room temperature for 1 min, then centrifuge at 6000 x g
for 1 min.
28. Add your DNA to a clean, WELL-LABELED tube (e.g. Genomic DNA,
your initials, the date).
29. Get a clean 1.5ml tube and, with a P100 or P200 pipette, add 49ul of
water. Then, with a P2 or P10 pipette, add 1ul of your genomic DNA. Vortex
the sample and quick spin in the centrifuge.
30. With a P100 or P200, add the 50ul of your sample from #29 to a
uvette (cuvette). Take it to the Biophotometer to get a quantitative reading.
WRITE DOWN THE QUANTITY OF DNA IN micrograms/microliter

Prepared by: Dr. K. L. Hinkle

Associate Professor of Biology
Norwich University

WRITE DOWN THE QUALITY OF DNA by taking the 260/280nm ratio

Protocol #2: Amplifying a portion of the TAS2R38 gene by PCR

In this part of the experiment, you will amplify a region of your own TAS2R28
gene that contains one of the SNPs that determines sensation of bitter taste
phenotype. You have isolated your genomic DNA, and providing primers for
the TAS2R28 gene that flank the SNP in question, we will be able to amplify a
small fragment of this gene and then via restriction enzyme cutting,
determine which genetic allele you harbor.
Examine the following diagram to analyze the basis behind the SNP
within the TAS2R38 gene and how the genetic alteration can be
determined via restriction enzyme cutting.

Prepared by: Dr. K. L. Hinkle

Associate Professor of Biology
Norwich University

Polymerase Chain Reaction:

Polymerase Chain Reaction (PCR) is a commonly utilized biomedical
technique that allows amplification of a specific DNA sequence of choice. By
designing PCR primers that flank the region of DNA that you wish to
amplify, you can make thousands of DNA copies of the fragment of interest.
A PCR reaction contains three major steps: 1) Denaturing (separating the
strands of DNA with heat) 2) Annealing (cooling down the reaction so primers
anneal to the single stranded DNA templates, and 3) Synthesis (heating up
the reaction again so that DNA polymerase can amplify the region of
interest). This cycle of Denaturing/Annealing/Synthesis is repeated many
times (~30) so that by the time a PCR is over, a segment of DNA has been
8

Prepared by: Dr. K. L. Hinkle

Associate Professor of Biology
Norwich University

amplified to millions of copies. This amplification enables researchers to

further utilize the DNA to detect mutations, to subclone into vectors, or for
other purposes.
We will be amplifying a small segment of the TAS2R38 gene that contains the
SNP that distinguishes tasters from non-tasters in terms of bitter taste.
____ 1. Label one 0.2ml tube with your initials.
____ 2. With a P100 or P200 pipette, Add 45ul of the PCR Mix into your 0.2ml

tube. The PCR mix contains the following reagents (already prepared
for you):
REAGENT
1 REACTION
PCR buffer
5
50mM MgCl2
3
10mM dNTPs
1
PCR water
33.8
10uM your forward
1
primer
10uM your reverse
1
primer
Platinum Taq
0.2
Polymerase
____ 3. Pipet 5ul of your isolated GENOMIC DNA into your tube.
____ 4. Close the tube tightly, vortex briefly, and then quick spin (using

adapters in the centrifuge)

5.
Add your "PCR" tube to the thermocycler:
____
Thermocycler is programmed for:
Step 1: 94C for 3 minutes
Step 2: 94C for 45 seconds (denaturing)
Step 3: 64C for 45 seconds (annealing)
Step 4: 72C for 90 seconds (synthesis)
Step 5: GOTO Step 2 x 30
Step 6: :72C for 10 min
Step 7:Hold on ice (4C)

Protocol #3: Cutting your PCR fragment with restriction enzyme

HaeIII
Examine the figure previously shown demonstrating the SNP within the
TAS2R38 gene. Note that in some people, their DNA has a G in a specific
spot within the TAS2R38 gene whereas others have a C in that same spot.
This ONE nucleotide change causes a slight change ultimately in a taste
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Prepared by: Dr. K. L. Hinkle

Associate Professor of Biology
Norwich University

receptor protein, resulting in differences in taste perception. But how can we

tell whether or not you have the taster SNP or the non-taster SNP?
Restriction enzymes! Technically called restriction endonucleases, these
are bacterial enzymes (NOT found in humans!) that serve to cut DNA at very
specific sequences. This is a protective mechanism for bacteria so that they
can basically shred DNA that is not their own. HOWEVER, in biomedical
science, we manipulate these enzymes and use them for our benefit, as each
specific enzyme cuts at a VERY SPECIFIC DNA sequence. For example, the
enzyme HaeIII, which we are using today, cuts ONLY at DNA sequences
GGCC. And the enzyme cuts this sequence right down the middle (between
the center G and C). GGCC is the taster sequence, so someone who has
this DNA sequence and isolates their genomic DNA and amplifies the
TAS2R38 gene via PCR and subjects that product to HaeIII will get a cut
piece of DNA. However, a person that has the SNP GGGC in that same
position (substitute the central C for another G) will NOT be recognized or cut
by HaeIII. Therefore, the PCR product will remain intact and uncut. These
differences can be distinguished on an agarose gel later on that separates
____
fragments out by size.
1. Label one fresh 0.2ml tube U for undigested AND your initials.
Label another fresh 0.2ml tube with D for digest AND your initials.
____ 2. With a P20 or P200, add 20ul of your PCR product to the U tube.

3. With a P20 or P200, add 20ul of your PCR product to the D tube.

____ 4. With a P10, to the D tube only, add 1ul of HaeIII.

5. With a P10 or P20, to the D tube only, add 3ul of 10X buffer.
____ 6. With a P10 or P20, to the D tube only, add 6ul of water.
____ 7. Vortex the D tube. Add both the D and the U tube to the
thermocycler and incubate at 37C for 30 minutes.
____
____

Protocol #4: Gel electrophoresis of Restriction Enzyme Digested

TAS2R38 PCR Products
Agarose gel electrophoresis is utilized to separate out DNA
fragments/molecules by SIZE. The larger the fragment, the less far it will run
through the gel; conversely, the smaller the fragment, the further it will run
through the gel. This allows us to visualize specific fragments of DNA (e.g.
PCR products) in order to determine whether an experiment was successful.
Agarose is a polysaccharide polymer that forms structures ("fibers") with
other agarose polymers, forming a rigid structure with pores. At higher
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Prepared by: Dr. K. L. Hinkle

Associate Professor of Biology
Norwich University

temperatures, agarose can be found in liquid form; however, as agarose

cools, it will solidify but still maintain "pores" that DNA or RNA fragments can
run through.
In order for nucleic acid to run through the gel, a charge is applied
through the electrophoresis chamber, from the ANODE to the CATHODE.
DNA is negatively charged due to the phosphate on the sugar backbone;
therefore, the DNA will migrate toward the POSITIVE electrode (cathode) due
to positive/negative attraction.
To visualize fragments or subunits of nucleic acid in an agarose gel,
one must apply a fluorescent indicator/dye that binds to double stranded or
complexed DNA/RNA. In this experiment, we will use a stain called SybrSafe.
SybrSafe is a cyanine compound ((Z)-4-((3-Methylbenzo[d]thiazol-2(3H)ylidene)methyl)-1-propylquinolin-1-ium 4-methylbenzenesulfonate) that
binds to DNA and, when bound to DNA, absorbs uv light and emits green
light. Without the dye in the gel, the DNA would be invisible; with the dye in
the gel and exposure on a uv light box, we will be able to see our DNA
products of interest.
Also important in any gel is to have a SIZE STANDARD. Since we are
looking for products at specific sizes, we need to have a reference with
KNOWN sizes so that we can compare our unknowns to the known
fragments.
Undigested TAS2R38 PCR fragment OR digested with G SNP :
221 bp fragment
Digested TAS2R38 PCR fragment if C SNP: 177bp and 44bp
fragments

In each gel, DNA ladder will be added to lane #1.

____1. With a P10 or P20, add 15ul of your U (undigested) DNA to one of
the lanes on the gel.

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Prepared by: Dr. K. L. Hinkle

Associate Professor of Biology
Norwich University

____2. With a P10 or P20, add 15ul of your D (digested) DNA to the next
lane of the gel. Make sure you record which lanes of which gel you
have added DNA to!
____3. When all students have added their DNA to the gel, electrophoresis
will start and we will run the gels for 30 minutes.
____4. Examine the gel under UV light. Determine whether your D digested
band is one 221bp band or two bands at 177bp and 44bp. You may also have
three bands (why might this be?)
Based on the GENOTYPE results, formulate a hypothesis about
whether you are a bitter TASTER or NON-TASTER.

Are you homozygous TASTER? Homozygous NON-TASTER? Or

Heterozygous taster/non-taster?

FINAL WRAP-UP
We have tested your genotype. Now lets test your PHENOTYPE related to
that genotype.
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Prepared by: Dr. K. L. Hinkle

Associate Professor of Biology
Norwich University

____1. Take a piece of PTC paper and set it on your tongue.

____2. Can you taste the PTC paper? ______________
____3. Does this make you a TASTER or NON-TASTER?
____4. How does this match up with your GENOTYPE?
____5. Based on your own genotype, predict what genotypes your
birth mother and birth father had. (Think Mendelian genetics!)

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