Download Using a Single-Nucleotide Polymorphism to Predict

Moorpark College STEP Workshops
Using a Single-Nucleotide Polymorphism
to Predict Bitter-Tasting Ability
(adapted from a Dolan DNA Learning Center Protocol)
Introduction
The ability to taste what you are eating is dependent on a series of specialized
cells on the surface of the tongue that act as chemical receptors. These taste receptors
detect one of the five basic tastes: sweet, sour, bitter, salty, and umami (glutamate).
When you eat something sour, molecules in the food bind to specific receptors on the
surface of taste cells that detect sour taste. Once the receptors are activated, these
cells send nervous impulses to the regions of the brains that deal with taste, and you
perceive a sour taste. Differ types of receptors are activated by different chemical
compounds, and the impulses they send to the brain are interpreted as other tastes.
Bitter is another of the five basic tastes. Bitter-tasting compounds are often quite
toxic to mammals, and the ability to detect these compounds and avoid foods that
contain them is of benefit to survival. Bitter-tasting compounds are recognized by a
large family of receptors that are encoded by about 30 different genes. One of these
genes, TAS2R38, was identified in 2003. This gene codes for a G-protein coupled receptor
(GPCR) that is activated by the chemical Phenylthiocarbamide (PTC). When molecules of
PTC bind to the TAS2R38 receptor some people perceive an extremely bitter taste, but for
other people the substance is completely tasteless. Subsequent studies by Albert
Blakeslee (1932), at the Carnegie Department of Genetics, showed that the inability to
taste PTC is a recessive trait that varies in the human population. Tasters versus nontasters could be detected by a simple chemical test, but the molecular basis for these
different phenotypes remained unknown until the TAS2R38 gene was discovered and
sequenced (Kim, et al. 2003).
The DNA sequence from the TAS2R38 gene varies at five positions within the human
population, and in each case the variable position is only a single base pair long. This type
of polymorphism within a DNA sequence is termed a Single Nucleotide Polymorphism
(SNP). Although there are five different SNPs within the TAS2R38 gene, only three of
these variants strongly influence bitter taste perception. The three SNPs that influence
bitter taste perception are listed in Table 1. Population level studies indicate that the
three "bitter taste" SNPs are inherited as a unit, which is referred to as a haplotype.
The "taster" haplotype has the amino acids proline(49), alanine(261) and valine(296)
(PAV), while the nontaster haplotype has the amino acids alanine(49), valine(261) and
isoleucine(296) (AVI) in the same positions. (The positions of these three SNPs are
indicated on the aligned polypeptide sequence in Appendix 1.)
In this experiment you will determine your genotype by PCR followed by a
restriction digest. You will use these results to predict whether you will be able to taste
PTC, and you will test your prediction by tasting PTC paper. Your results link molecular
genetics to Mendelian inheritance, and demonstrate how SNPs can be used can be used as
molecular markers to predict phenotypes.
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Moorpark College STEP Workshops
Nucleotide
Position
TASTER
NONTASTER
Codon
Amino Acid
Codon
Amino Acid
145
CCA
Proline
GCA
Alanine
785
GCT
Alanine
GTT
Valine
886
GTC
Valine
ATC
Isoleucine
Table 1. SNPs in the TAS2R38 gene that are associated with the ability to
taste Phenylthiocarbamide (PTC). The position refers to the base-pair in the
DNA sequence where the polymorphism occurs. The nucleotide substitution
and the resulting amino acid are also shown. All three SNPs constitute a
haplotype that is usually inherited as a single unit.
March 28, 2009
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Protocols
Collecting and Isolating DNA from Cheek Cells
This procedure is used to isolate DNA from human cheek cells. These cells are
dislodged from the inner surface of one of your cheek pouches by gentle scrapping with
the blunt end of a sterile flat toothpick. The harvested cells are then transferred to a
tube of isotonic saline solution to help remove contaminants (saliva and food particles).
After a brief rinse, the cells are pelleted by centrifugation. After pouring off the saline
solution, the cells are resuspended in an aqueous mixture containing Chelex® resin. The
samples are then boiled to rupture the cell membranes and extract the DNA. As the cells
lyse they also release enzymes that degrade nucleic acids (DNA and RNA), but many of
these enzymes require metal ions as cofactors (Mg++). The Chelex® resin in the sample
tubes binds all the free metal ions present in the samples, and this inactivates any
enzymes that require these cofactors. Other enzymes not inhibited by the Chelex® are
heat denatured during the boiling process. This simple extraction procedure yields DNA of
sufficient quality to perform PCR, if the samples are used relatively soon (within a few
days). If samples need to be stored for longer periods of time additional purification
should be carried out to improve the archival quality of the DNA.
_____ 1.
Obtain a 1.5 ml microfuge tube of sterile 0.9% saline solution.
_____ 2.
Write your initials on the top of the microfuge tube of saline solution
with one of the permanent waterproof markers provided.
_____ 3.
Gently scrape the inner surface of your cheek a thoroughly a few times
with the blunt end of a sterile flat toothpick. Stick the blunt end of the
toothpick in the saline solution and vigorously stir the solution with the
toothpick to dislodge the cells. Place the used toothpicks in the waste
container.
_____ 4.
Repeat the previous step two more times, so that you have collected
and transferred three samples of cheek cells to your tube of saline
solution. (This insures that you have enough cells to provide an
adequate amount of DNA for PCR.) Place the used toothpicks in waste
container.
_____ 5.
Briefly vortex your tube of saline solution for 5 - 10 seconds to breakup
any lumps of cells.
_____ 6.
Place your sample tube, along with other samples, in a microcentrifuge.
(Make sure the microcentrifuge is balanced.) Centrifuge the samples at
full speed for 90 seconds.
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_____ 7.
Remove your tube of saline solution from the centrifuge and visually
inspect the bottom of the tube for a small pellet of cells. (You must be
able to see a pellet to insure a sufficient cell sample.) After locating
your cell pellet carefully pour off the supernatant into a suitable waste
container. Be carefully not to disturb or lose your cell pellet.
_____ 8.
Obtain a 1.5 ml microfuge tube of Chelex®. Vigorously shake the Chelex®
to resuspend the resin and quickly pour the entire contents into the
empty saline tube containing your cell pellet. Tap out any remaining
Chelex® into the cell pellet tube.
_____ 9.
Resuspend the cells and mix them with the Chelex® resin by briefly
vortexing the tube for 5 - 10 seconds. Repeat this step until the cell
pellet has been dislodged from the wall the microcentrifuge tube, and
has broken up.
_____ 10.
Place the sample tube in a floating microfuge tube rack, along with other
student sample tubes, and place the tube rack in boiling water for 10
minutes.
_____ 11.
After boiling, remove your tube from the tube rack and vigorously shake
the sample tube for 5 seconds.
_____ 12.
Place your tube, along with other student samples, in a balanced
configuration in a microcentrifuge and spin for 90 seconds at full speed.
_____ 13.
Store on ice until you are ready to continue with the next step.
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Amplifying DNA by PCR
The Polymerase Chain Reaction (PCR) is a technique for producing a large number
of copies of a specific region of DNA. (DNA amplification). PCR employs a thermal stable
DNA polymerase (Taq), isolated from a species of bacteria found in thermal pools, to
replicate the target DNA during a series of successive heating and cooling cycles (thermal
cycles). The Ready-To-GoTM PCR Beads used in this protocol contain lyophilized buffer,
thermal stable DNA polymerase, deoxyribonucleotides triphosphates (dNTPs -- dATP, dCTP,
dGTP, dTTP), and MgCl2. When these Ready-To-GoTM PCR Beads are dissolved in an
appropriate volume of water containing a specific pair of single strand-oligonucleotide
primers (PTC mix), all of the reagents required for PCR are present except the template
DNA. This is usually added to the reaction mixture last to minimize nonspecific
amplification prior to thermal cycling.
During thermal cycling heat is used to denature the double strand-DNA template.
After the DNA is denatured, the reaction mix is cooled to a temperature low enough to
allow the ssDNA primers to anneal to their complementary sites on the denatured
template DNA. Once the primers have annealed, the temperature is raised slightly, and
the DNA polymerase adds nucleotides to the 3' ends of the primers, thereby producing the
complementary strand of DNA. These steps (denature, anneal, extend) are then repeated
over and over again, and after every cycle the number of copies of DNA delimited by the
primers is doubled.
_____ 1.
Obtain a 0.2 ml PCR tube containing a Ready-To-GoTM PCR Bead. (Try to
handle only the PCR tube you use.) Check to make sure your bead is at the
bottom of the tube before you open it.
_____ 2.
Use a P200 micropipet fitted with a fresh tip to add 22.5 µl of PTC
Primer/loading dye mix to your PCR tube. (Touch the end of the pipet tip to
the inner wall of the PCR tube some distance above the bead and add the
PTC mix. Allow the PTC mix to flow down to the bead. Don't touch the tip
to the bead.)
_____ 3.
Use a P20 micropipet fitted with a fresh tip to add 2.5 µl of your cheek cell
DNA directly to the PCR mix.
_____ 4.
Briefly centrifuge your PCR tube in a Picofuge for a few seconds to insure the
reaction mix is at the bottom of the tube.
_____ 5.
Locate an empty square on the PCR grid sheet and write your name in this
square. Find the corresponding location in the PCR rack on ice and insert
your tube. (Do not forget to sign out your location.)
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Restriction Digest of PCR Products with HaeIII
Once the target DNA has been amplified we still need a method of determining
whether the Single Nucleotide Polymorphism (SNP) is present in the PCR products.
(Remember, what we are looking for is a single base pair difference (C versus G) in a
fragment of DNA 221 base-pairs long.) We could sequence our PCR products to determine
what base is present at the polymorphic site, but there is an easier way to obtain the
same information. A restriction enzyme (an endonuclease) is capable of scanning a
double-strand DNA molecule for a specific sequence of base pairs called a restriction site.
If a restriction site is present the dsDNA is cleaved by the restriction enzyme within the
restriction site. If there is no restriction site for the specific enzyme the dsDNA is not
cut.
The restriction enzyme HaeIII has a 4 base long restriction site "GGCC". This
restriction site is present at positions 43-46 in those PCR products with a cysteine (C) at
position 45, but it is absent in the others with a guanine at this position (G). In the case
of the former, HaeIII cuts the PCR product into two fragments, in the case of the latter
the PCR product is not cut. After incubating the PCR products with HaeIII, the cut and
the uncut PCR products can be separated on the basis of size. Since we are dealing with
millions of copies of our target DNA, this cut or uncut DNA can be stained and visualized.
_____ 1.
Obtain two 1.5 ml microfuge tubes. Using a permanent waterproof
markers label one tube "U" (for undigested) and the other tube "D" (for
digested). Put your initials on both tubes so you will be able to relocate
them later.
_____ 2.
Using a P20 micropipette with a fresh tip, transfer 10 µl of your PCR product
to the "U" tube. Store this tube on ice until you are ready to begin gel
electrophoresis.
_____ 3.
Using a fresh tip, transfer the remaining 15 µl of your PCR product to your
"D" tube.
_____ 4.
Using a fresh tip, add 1 µl of HaeIII restriction enzyme directly into the PCR
product in tube "D". Be sure to immediately return the restriction enzyme
to the ice bucket or cold block when you are finished with it.
_____ 5.
Briefly centrifuge your tube "D" to pool the reagents at the bottom of the
tube.
_____ 6.
Place your tube "D", along with other student samples, in a 37º C water bath.
Allow all the samples to undergo digestion for 30 - 60 minutes.
_____ 7.
After the digestion is complete remove the tubes from the water bath and
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Moorpark College STEP Workshops
store on ice or freeze until you are ready to begin electrophoresis.
Analysis of the PCR Restriction Digests by Gel Electrophoresis
Gel electrophoresis is a technique that can be use to separate DNA fragments that
differ in size. The sample DNA is loaded into depressions (wells) in an agarose gel that
has been submerged in running buffer. An electrical current passes through the running
buffer and the gel, thereby drawing the negatively charged DNA molecules through the
colloidal gel matrix toward the cathode. Larger molecules have more difficulty moving
through the gel and travel slower. Small molecules can slip between the particles that
make up the gel more easily and travel faster. As the sample progress through the gel the
fragments sort into distinct bands based on their size and mobility. After electrophoresis,
the bands of DNA can be visualized by staining the gel with a dye with a high affinity for
DNA.
_____ 1.
Set up as many 2% agarose gels as needed to run all of the digested and
undigested samples of the PCR products. (Each student will need two lanes.)
_____ 2.
Using a P20 micropipette with a fresh tip, load 20 µl of pBR322/BstNI size
markers into the far left well of each gel.
MARKER
pBR32
2/
BstNI
STUDENT 1
U
STUDENT 2
D
U
D
_____ 3.
Using a fresh tip, load 10 µl of sample from tube U (undigested PCR
product/dye mixture) to the lane assigned to you for your sample "U".
_____ 4.
Using a fresh tip, load 16 µl of sample from tube D (digested PCR
product/loading dye mixture to the lane assigned to you for your sample "D".
_____ 5.
Once the gel is completely loaded, run the gel at 130 V for approximately 30
minutes. (Adequate separation will have occurred when the cresol red dye
front has moved at least 50 mm from the wells.)
_____ 6.
View the gel using a transilluminator, and photograph the resulting bands
with a digital camera.
tt
TT
Tt
_____ 7.
Determine your genotype
and record your results.
U
D
U
D
U
D
well
221
171
44
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Results
Observe the photograph of the stained gel containing your PCR digests and those
from the other students. Orient the photograph with the sample wells at the top and
compare your results to the others. The "U" lane contains your undigested PCR products.
These fragment are 221 base pairs long. The "D" lane contains your digested PCR
products.
If the "D" lane contains a single band, your PCR products were not cut by the
restriction enzyme HaeIII. In this case both copies of your TAS2R38 gene are identical
(the same allele). This form of the gene has a "G" at base-pair 45 in the DNA sequence,
and the change in the DNA sequence eliminates the restriction site for HaeIII (GGCC
becomes GGGC). As a result, none of the PCR fragments are cleaved. This SNP also
changes the amino acid at position 49 in the protein from proline to alanine, and it is
inherited along with the other two SNPs that form the AVI nontaster haplotype. The
resulting protein receptor is incapable of being bound by PTC, and, to put it simply, you
cannot detect the presence of PTC. Since both copies of the allele are identical, your
genotype is tt (homozygous nontaster).
If your "D" lane contains two bands, both copies of your TAS2R38 lack the
nucleotide substitution at base-pair 45. In this case, the restriction site for HaeIII (GGCC)
is present and the enzyme cuts the PCR products into two sets of fragments, 171 bp and
44 bp long. The protein coded from this allele has the normal amino acids at positions 49
(proline), 261 (alanine) and 296 (valine). The resulting protein receptor is capable of
being bound by PTC, and you are a taster. Since both copies of the allele are identical,
your genotype is TT (homozygous taster).
If your "D" lane contains three bands, you have two different alleles for the
TAS2R38 gene. An allele with a "G" at position 45 that is not cut by the restriction enzyme
(the nontaster allele), and an allele with a "C" at position 45 that is cut by the restriction
enzyme (the taster allele). You produce some normal receptors that can be bound by
PTC, and some aberrant receptors that cannot. Since fewer receptors are being bound by
PTC, fewer impulses are sent to the brain, and your brain interprets the reduced signal as
a slightly bitter taste. Since the two alleles are different, your genotype is Tt
(heterozygous weak taster).
As a scientist, there comes a point in every experiment when you understand how
something works, but you are not sure if your explanation can be applied to the real
world. It's time to find out.
March 28, 2009
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Moorpark College STEP Workshops
Based on the results of your PCR digest make a prediction about whether you will be able
to taste PTC or not.
I think paper impregnated with PTC will taste like:
_____ damp paper (Nontaster t/t)
_____ damp, disgusting, slightly bitter paper (Weak taster T/t)
_____ YUK!!!! This is something my tongue never wants to meet again
(Strong taster TT)
Place one strip of control test paper on the center of your tongue for several seconds.
Note the taste. Then remove the control paper, and place a strip of PTC taste paper in
the center of your tongue for several seconds. How would you describe the taste of the
PTC paper, as compared to the control: strongly bitter; weakly bitter; no taste other than
paper.
Correlate the PTC genotypes with the phenotypes and record the class results in
the table that follows.
Phenotype
Genotype
Strong Taster Weak Taster Nontaster
TT (homozygous)
Tt (heterozygous)
tt (homozygous)
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Moorpark College STEP Workshops
Some Questions to think about.
1. According to your class results, how well does the TAS2R38 genotype predict the PTCtasting phenotype?
2. What does this tell you about classical Mendelian dominant/recessive pattern of
inheritance? Can we distinguish the homozygous dominant phenotype (TT) form the
heterozygous phenotype (Tt)?
3. Could these results allow us to predict how likely a person is to avoid or ingest other
substances that contain bitter tasting compounds?
4. Does this experiment have any practical application in the real world?
5. Can you explain how it's possible for two parents, who are both tasters, to produce a child
that is a nontaster? Can you calculate the probability of such an event?
6. If the filled symbols are tasters and the unfilled symbols are nontasters, what are the
genotypes of the individuals in the two pedigrees shown above? (Squares = males, circles =
females). (If you can't determine the genotype for any individual, list all the possible
genotypes.)
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Additional Readings
Molecular Evolution
Kim et al. (2003) sequenced the PTC gene in 6 primate species (including humans) to
determine the original form of the PTC gene. All nonhuman primates were homozygous for the PAV
form, which indicates that the AVI form arose in humans after they diverged from the nearest
common primate ancestors. Five different haplotypes were observed worldwide. In Europeans and
Asians, the taster haplotype PAV and the nontaster haplotype AVI made up the vast majority of
haplotypes present. Two additional haplotypes, PVI and AAI, were observed only in individuals of
sub-Saharan African ancestry, which is consistent with other reports of increased gene haplotype
diversity in this population. The common nontaster AVI haplotype was observed in all populations
except southwest Native Americans, who were exclusively homozygous for the PAV haplotype,
consistent with the reported low frequency of nontasters in this population. Thus, overall, the
worldwide distribution of these haplotypes is consistent with the large anthropological literature on
the distribution of this phenotype.
Molecular Genetics
Mangold et al. (2008) presented evidence suggesting that the 3 SNPs in the TAS2R38 gene
identified by Kim et al. (2003) may play a role in the development of nicotine dependence among
African Americans. The taster haplotype PAV was inversely associated (p = 0.0165), and the
nontaster haplotype AVI was positively associated (p = 0.0120), with smoking quantity in a study of
1,053 African American smokers. The nontaster haplotype was positively associated with all
measures of nicotine dependence in female African American smokers (p = 0.01-0.003). No
significant associations were observed in a sample of 515 European smokers. Mangold et al. (2008)
postulated that heightened oral sensitivity confers protection against nicotine dependence.
Blakeslee, A. F. 1932. Genetics of sensory thresholds: Taste for phenylthiocarbamide. 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., Drayna, D. 2003. Positional cloning of
the human quantitative trait locus underlying taste sensitivity to phenylthiocarbamide.
Science 299: 1221-1225, 2003.
Mangold, J. E., Payne, T. J., Ma, J. Z., Chen, F., Li, M. D. 2008. Bitter taste receptor gene
polymorphisms are an important factor in the development of nicotine dependence in
African Americans. J. Med. Genet. 45: 578-582, 2008.
Mueller, K. L., Hoon, M. A., Erlenbach, I., Chandrashekar, J., Zukes, C. S., 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: 423427.
March 28, 2009
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Moorpark College STEP Workshops
(Appendix 1) TAS2R38 Amino Acid Alignment
145
CCA (PRO) → GCA (ALA)
HS+
PT
PP
GG
PY
HK
mltltrirtv
..........
..........
..........
..........
.......ca.
.......c..
syevrstflf
..........
..........
..........
..........
..........
..........
isvlefavgf
..........
..........
..........
..........
..........
..........
ltnafvflvn
..........
..........
..........
..........
.....i....
.....i....
fwdvvkrqal
........p.
........p.
........p.
........p.
........p.
........p.
snsdcvllcl
..........
..........
..........
..........
..........
..........
HSHS+
PT
PP
GG
PY
HK
sisrlflhgl
..........
..........
..........
..........
..........
..........
lflsaiqlth
..........
..........
..........
..........
..........
..........
fqklseplnh
..........
..........
..........
..........
..........
..........
syqaiimlwm
..........
..........
..........
..........
........xi
..........
ianqanlwla
..........
..........
..........
..........
..x.......
..........
aclsllycsk
..........
..........
..........
..........
..........
t.........
HSHS+
PT
PP
GG
PY
HK
lirfshtfli
..........
..........
..........
..........
..........
..........
claswvsrki
..........
..........
..........
..........
..........
..........
sqmllgiilc
..........
..........
..........
..........
...x......
c.........
scictvlcvw
..........
..........
..........
..........
..........
..........
cffsrphftv
..........
..........
..........
..........
..........
.y........
ttvlfmnnnt
..........
..........
..........
..........
..........
..........
HSHS+
PT
PP
GG
PY
HK
rlnwqikdln
..........
..........
..........
..........
..........
..........
lfysflfcyl
..........
..........
..........
..........
..........
..........
wsvppfllfl
..........
..........
..........
..........
..........
..........
vssgmltvsl
..........
..........
..........
..........
..........
..........
grhmrtmkvy
..........
..........
..........
..........
..........
..........
trnsrdpsle
..........
..........
..........
..........
..........
...f......
HSHS+
PT
PP
GG
PY
HK
ahikalkslv
..........
..........
..........
..........
..........
..........
sffcffviss
..........
..........
..........
..........
..........
..........
cvafisvpll
.a........
.a........
.a........
.a........
.a........
.a........
ilwrdkigvm
..........
..........
..........
..........
....n.....
..........
vcvgimaacp
..........
..........
..........
..........
..........
..........
HSHS+
PT
PP
GG
PY
HK
naklrravmt
..........
........t.
........t.
........t.
........t.
........t.
illwaqsslk
..........
..........
..........
..........
........x.
..........
vradhkadsr
..........
..........
..........
..........
..........
..........
tlc
...
...
...
.p.
.l.
.l.
HS-
785 GCT (ALA) → GTT (VAL)
March 28, 2009
886 GTC (VAL) → ATC (ISO)
Page 12 of 13
sghaailisg
.....v....
.....v....
.....v....
.....v....
.....v....
.....v....
Homo sapiens NP_789787 non taster
Homo sapiens NP_789787 taster
Pan troglodytes AAS67622
Pan paniscus AAS67621
Gorilla gorilla AAS67623
Pongo pygmaeus AAS67624
Hylobates klossii AAS67625
Predicting Bitter-Tasting Ability
Moorpark College STEP Workshops
Homo sapiens NP_789787
1
61
121
181
241
301
mltltrirtv
sisrlflhgl
lirfshtfli
rlnwqikdln
ahikalkslv
naklrravmt
syevrstflf
lflsaiqlth
claswvsrki
lfysflfcyl
sffcffviss
illwaqsslk
isvlefavgf
fqklseplnh
sqmllgiilc
wsvppfllfl
caafisvpll
vradhkadsr
ltnafvflvn
syqaiimlwm
scictvlcvw
vssgmltvsl
ilwrdkigvm
tlc
fwdvvkrqal
ianqanlwla
cffsrphftv
grhmrtmkvy
vcvgimaacp
snsdcvllcl
aclsllycsk
ttvlfmnnnt
trnsrdpsle
sghaailisg
isvlefavgf
fqklseplnh
sqmllgiilc
wsvppfllfl
caafisvpll
vradhkadsr
ltnafvflvn
syqaiimlwm
scictvlcvw
vssgmltvsl
ilwrdkigvm
tlc
fwdvvkrqpl
ianqanlwla
cffsrphftv
grhmrtmkvy
vcvgimaacp
snsdcvllcl
aclsllycsk
ttvlfmnnnt
trdsrdpsle
sghaavlisg
Pan troglodytes AAS67622
1
61
121
181
241
301
mltltrihtv
sisrlflhgl
lirfshtfli
rlnwqikdln
ahikalkslv
naklrravtt
syevrstflf
lflsaiqlth
claswvsrki
lfysflfcyl
sffcffviss
illwaqsslk
Pan paniscus AAS67621
1 mltltrihtv syevrstflf isvlefavgf ltnafvflvn fwdvvkrqpl snsdcvllcl
61
121
181
241
301
sisrlflhgl
lirfshtfli
rlnwqikdln
ahikalkslv
naklrravtt
lflsaiqlth
claswvsrki
lfysflfcyl
sffcffviss
illwaqsslk
fqklseplnh
sqmllgiilc
wsvppfllfl
caafisvpll
vradhkadsr
syqainmlwm
scictvlcvw
vssgmltvsl
ilwrdkigvm
tlc
ianqanlwla
cffsrphftv
grhmrtmkvy
vcvgimaacp
aclsllycsk
ttvlfmnnnt
trdsrdpsle
sghaavlisg
isvlefavgf
fqklseplnh
sqmllgiilc
wsvppfllfl
caafisvpll
vradhkadsr
ltnafvflvn
syqaiimlwm
scictvlcvw
vssgmltvsl
ilwrdkigvm
tpc
fwdvvkrqpl
ianqanlwla
cffsrphftv
grhmrtmkvy
vcvgimaacp
snsdcvllcl
aclsllycsk
ttvlfmnnnt
irdsrdpsle
sghaavlisg
isvlefavgf
fqklseplnh
sqmxlgiilc
wsvppfllfl
caafisvpll
vrabhkadsr
ltnafiflvn
syqaiimlxi
scictvlcvw
vssgmltvsl
ilwrnkigvm
tlc
fwdvvkrqpl
iaxqanlwla
cffsrphftv
grhmrtmkvy
vcvgimaacp
snsdcvllcl
aclsllycsk
ttflfmnnnt
trdsrdpsle
sghaavlisg
Gorilla gorilla AAS67623
1
61
121
181
241
301
mltltrirtv
sisrlflhgl
lirfshtfli
rlnwqikdln
ahikalkslv
naklrravtt
syevrstflf
lflsaiqlth
claswvsrki
lfysflfcyl
sffcffviss
illwaqsslk
Pongo pygmaeus AAS67624
1
61
121
181
241
301
mltltricav
sisrlflhgl
lirfshtfli
rlnwqikdln
ahikalkslv
naxlrravtt
syevrstflf
lflsaiqlth
claswvsrki
lfysflfcyl
sffcffviss
illwaqssxk
Hylobates klossii AAS67625
1 mltltrictv syevrstflf isvlefavgf ltnafiflvn fwdvvkrqpl snsdcvllcl
61
121
181
241
301
sisrlflhgl
lirsshtfli
rlnwqikdln
ahikalkslv
naklrravtt
March 28, 2009
lflsaiqlth
claswvsrki
lfysflfcyl
sffcffviss
illwaqsslk
fqklseplnh
cqmllgiilc
wsvppfllfl
caafisvpll
vradhkadsr
syhaiimlwm
scictvlcvw
vssgmltvsl
ilwrdkigvm
tlc
Page 13 of 13
ianqanlwla
cyfsrphftv
grhmrtmkvy
vcvgimaacp
tclsllycsk
ttvlftnnnt
trdfrdpsle
sghaailisg
Predicting Bitter-Tasting Ability