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Using a Single-Nucleotide
Polymorphism to Predict
Bitter-Tasting Ability
1. The TAS2R38 protein has not been crystallized and there is no associated PDB number.
(TAS2R38=PTC gene)
2. The nucleotide and putative amino acid sequence of the TAS2R38 gene is known. The
GenBank Accession # is NM_176817.
3. However the structure of the receptor and its ligand-binding has been predicted by
computer modeling. The reference is
Modeling the human PTC bitter-taste receptor interactions with bitter tastants.
Floriano WB, Hall S, Vaidehi N, Kim U, Drayna D, Goddard WA 3rd.
J Mol Model. 2006 Sep;12(6):931-41.
!
Using restriction enzymes to cut DNA
Restriction Enzymes: cut covalent
phosphodiester bonds at restriction /
recognition sites (4 - 8 nucleotide sequence,
often a palindrome); often in staggered fashion
---> restriction fragments w/ “sticky ends”. !
Restriction site
DNA 5!
3!
3!
5!
G AAT T C
C T TAA G
Restriction enzyme cuts
the sugar-phosphate
backbones at each arrow
G
Naming: genus, species, strain, # discovered !
C T TAA
AAT T C
G
Sticky end
ex. ECoRI, HindIII!
AAT T C
DNA fragment from
another source is added.
Base pairing of sticky
ends produces various
combinations.
G AAT T C
C T TAA G
G
C T TAA
G
Fragment from different
DNA molecule cut by the
same restriction enzyme
G AAT T C
C T TAA G
One possible combination
DNA ligase
seals the strands.
Recombinant DNA molecule
2!
http://www.dnai.org/b/index.html - Manipulation / Techniques/ Cutting & Pasting
!
Gel Electrophoresis!
www.dnai.org/index.htm Manipulation / Sorting & sequencing!
Digest with restriction enzymes!
Concentration of agarose gel can be
increased for finer separation!
DNA = negatively charged
smaller fragments travel faster &
therefore farther
correlate distance to size
http://www.phschool.com/science/biology_place/labbench/lab6/concepts2.html!
SNP’S & RFLP’S!
SNP’s (single nucleotide polymorphisms) occur at a frequency !
43!
of about 1 in every 1,000 nucleotides. Although some have a !
biological effect on the individual, most have no effect. However, !
they may be used as genetic markers in order to locate genes that !
cause or predispose to disease or influence other traits.!
34!
If you pick a restriction enzyme that recognizes the sequence TTAAA and !
cuts it between the T and A, then person 1’s DNA will be cut !
differently than person 2’s DNA.!
PERSON 1!
TTAAATCGTGCTGATATTGGCGATGATCGGGGGTTTAAACCGCTA!
PERSON 2!
TTAAATCGTGCTGATATTGGCGATGATCGGGGGTTTGAACCGCTA
!
!
!
!
!
!
!
!
RFLP’s - restriction fragment length polymorphisms ---> !
9!
2!
!
!
!
!
!
!
!
!
!
person 1
!!
!!
!2!
genetic markers (certain pattern of RFLP’s always associated !
with a particular disorder/trait)!
!
!
!
!!
Person 1 will have different length fragments cut by the restriction enzyme than person 2. If you analyze their DNA by gel
electrophoresis, you will get different patterns due to the different length fragments of DNA ((RFLP’s).!
Person 1 - fragment lengths of 2, 9 , 34
Person 2 - fragment lengths of 2, 43!
!
We will assume that each person has 2 of the same alleles for this trait. Two pieces of DNA of the same length stop on the gel at
the same place and still appear as a single band. What would the banding pattern look like for someone that was heterozygous?
Hint: fragment lengths = 2,9,34,43!
RFLP’s - restriction fragment length
polymorphisms ---> genetic markers :certain
pattern of RFLP’s always associated with a
particular disorder/trait.
!!
SNP’s - single nucleotide polymorphisms;
occur at a frequency of about 1 in every
1,000 nucleotides. Although some have a
biological effect on the individual, most
have no effect. However, they may be
used as genetic markers in order to locate
genes that cause or predispose to disease
or influence other traits.!
Haplotype - a set of single nucleotide
polymorphisms (SNPs) on a single
chromatid that are statistically associated.
This information is very valuable for
studying similarities and differences in
humans and investigating the genetics
behind common diseases - see International HapMap Project. http://
www.hapmap.org/thehapmap.html.en
(http://en.wikipedia.org/wiki/Haplotype)
DNA in the Cell!
chromosome!
cell nucleus!
Double stranded
DNA molecule!
Target Region for PCR!
Individual
nucleotides!
PCR - Polymerase Chain Reaction!
**use!
.2 ml
tubes!
http://www.dnai.org/b/index.html - Manipulation/Amplifying!
http://learn.genetics.utah.edu/content/labs/pcr/ !
*Amplifies DNA segments to make unlimited quantities of
sections of DNA / genes of interest!
• DNA is heated, denatured to break hydrogen bonds!
• Cool and add primers, DNA polymerase is added and
DNA is synthesized!
• Repeat; Amount of DNA doubles with each cycle!
= forward primer!
= intentionally introduced
mismatch in primer!
The forward primer binds within the
TAS2R38gene, from nucleotides 101–144.
There is a single mismatch at position 143,
where the primer has a G and the gene has
an A. This mismatch is crucial to the PCR
experiment, because the A in the PTC
sequence is replaced by a G in each of the
amplified products. This creates the first G of
the HaeIII recognition sequence GGCC (this
is not naturally present in the
TAS2R38gene), allowing the amplified taster
allele to be cut. The amplified nontaster
allele reads GGGC and is not cut. !
221 bp
177 & 41 bp
9!
http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=607751!
12!
Using a Single-Nucleotide Polymorphism to Predict Bitter-Tasting Ability
5
STUDENT LAB INSTRUCTIONS
INTRODUCTION
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 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.
A serendipitous observation at DuPont, in the early 1930s, first showed
a genetic basis to taste. Arthur Fox had synthesized some
phenylthiocarbamide (PTC), and some of the PTC dust escaped into the
air as he was transferring it into a bottle. Lab-mate C.R. Noller complained
that the dust had a bitter taste, but Fox tasted nothing—even when he
directly sampled the crystals. Subsequent studies by Albert Blakeslee, at
the Carnegie Department of Genetics (the forerunner of Cold Spring
Harbor Laboratory), showed that the inability to taste PTC is a recessive
trait that varies in the human population.
Albert Blakeslee using a voting
machine to tabulate results of
taste tests at the AAAS
Convention, 1938. (Courtesy Cold
Spring Harbor Laboratory
Research Archives)
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. Sequencing identified three nucleotide
Copyright © 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.
Using a Single-Nucleotide Polymorphism to Predict Bitter-Tasting Ability
6
positions that vary within the human population—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.
pharmacogenomics !!
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 boiling with Chelex resin, which binds
contaminating metal ions. 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 not—producing a restriction fragment length polymorphism
(RFLP) that can be separated on a 2% agarose gel.
Each student scores his or her genotype, predicts their tasting ability, and
then tastes PTC paper. Class results show how well PTC tasting actually
conforms to classical Mendelian inheritance, and illustrates the modern
concept of pharmacogenetics—where a SNP genotype is used to predict
drug response.
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.
DEFINITION Homo sapiens taste receptor, type 2, member 38 (TAS2R38),
RefSeqGene on chromosome 7.
ACCESSION NG_016141
SOURCE Homo sapiens (human)
Summary: This gene encodes a seven-transmembrane G protein-coupled
receptor that controls the ability to taste glucosinolates, a
family of bitter-tasting compounds found in plants of the Brassica
sp. Synthetic compounds phenylthiocarbamide (PTC) and
6-n-propylthiouracil (PROP) have been identified as ligands for
this receptor and have been used to test the genetic diversity of
this gene. Although several allelic forms of this gene have been
identified worldwide, there are two predominant common forms
(taster and non-taster) found outside of Africa. These alleles
differ at three nucleotide positions resulting in amino acid
changes in the protein (A49P, V262A, and I296V) with the amino acid
combination PAV identifying the taster variant (and AVI identifying
the non-taster variant).
Copyright © 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.
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Using a Single-Nucleotide Polymorphism to Predict Bitter-Tasting Ability
7
LAB FLOW
I.
ISOLATE DNA BY SALINE MOUTHWASH
II. AMPLIFY DNA BY PCR
III. DIGEST PCR PRODUCTS WITH HaeIII
IV. ANALYZE PCR PRODUCTS BY GEL ELECTROPHORESIS
–
+
Using a Single-Nucleotide Polymorphism to Predict Bitter-Tasting Ability
8
METHODS
I.
ISOLATE DNA BY SALINE MOUTHWASH
Reagents (at each student station)
Supplies and Equipment
0.9% saline solution, 10 mL
10% Chelex®, 100 µL (in 0.2- or 0.5-mL PCR
tube)
Permanent marker
Paper cup
Micropipets and tips (10–1000 µL)
1.5-mL microcentrifuge tubes
Microcentrifuge tube rack
Microcentrifuge adapters
Microcentrifuge
Thermal cycler (or water bath or heat
block)
Container with cracked or crushed ice
Vortexer (optional)
1. Use a permanent marker to label a 1.5-mL tube and paper cup with
your assigned number.
2. Pour saline solution into your mouth, and vigorously rinse your cheek
pockets for 30 seconds.
3. Expel saline solution into the paper cup.
Your teacher may instruct you to
collect a small sample of cells to
observe under a microscope.
4. Swirl the cup gently to mix cells that may have settled to the bottom.
Use a micropipet with a fresh tip to transfer 1000 µL of the solution
into your labeled 1.5-mL microcentrifuge tube.
5. Place your sample tube, along with other student samples, in a
balanced configuration in a microcentrifuge, and spin for 90 seconds
at full speed.
Before pouring off supernatant,
check to see that pellet is firmly
attached to tube. If pellet is loose
or unconsolidated, carefully use
micropipet to remove as much
saline solution as possible.
6. Carefully pour off supernatant into the paper cup. Try to remove most
of the supernatant, but be careful not to disturb the cell pellet at the
bottom of the tube. (The remaining volume will reach approximately
the 0.1 mark of a graduated tube.)
Food particles will not resuspend.
8. Withdraw 30 µL of cell suspension, and add it to a PCR tube
containing 100 µL of Chelex®. Label the cap and side of the tube
with your assigned number.
The near-boiling temperature lyses
the cell membrane, releasing DNA
and other cell contents.
Alternatively, you may add the cell
suspension to Chelex in a 1.5-mL
tube and incubate in a boiling
water bath or heat block.
7. Set a micropipet to 30 µL. Resuspend cells in the remaining saline by
pipetting in and out. Work carefully to minimize bubbles.
9. Place your PCR tube, along with other student samples, in a thermal
cycler that has been programmed for one cycle of the following
profile. The profile may be linked to a 4°C hold program. If you are
using a 1.5-mL tube, use a heat block or boiling water bath.
Boiling step:
99°C
10 minutes
10. After boiling, vigorously shake the PCR tube for 5 seconds.
Copyright © 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.
Using a Single-Nucleotide Polymorphism to Predict Bitter-Tasting Ability
To use adapters, “nest” the sample
tube within sequentially larger
tubes: 0.2 mL within 0.5 mL within
1.5 mL. Remove caps from tubes
used as adapters.
9
11. Place your tube, along with other student samples, in a balanced
configuration in a microcentrifuge, and spin for 90 seconds at full
speed. If your sample is in a PCR tube, one or two adapters will be
needed to spin the tube in a microcentrifuge designed for 1.5-mL tubes.
12. Use a micropipet with a fresh tip to transfer 30 µL of the clear
supernatant into a clean 1.5-mL tube. Be careful to avoid pipetting
any cell debris and Chelex® beads.
13. Label the cap and side of the tube with your assigned number. This
sample will be used for setting up one or more PCR reactions.
14. Store your sample on ice or at –20°C until you are ready to continue
with Part II.
II. AMPLIFY DNA BY PCR
Reagents (at each student station)
Supplies and Equipment
*Cheek cell DNA, 2.5 µL (from Part I)
*PTC primer/loading dye mix, 22.5 µL
Ready-To-GoTM PCR beads (in 0.2-mL or
0.5-mL PCR tube)
Permanent marker
Micropipet and tips (1–100 µL)
Microcentrifuge tube rack
Thermal cycler
Container with cracked or crushed ice
Shared Reagent
Mineral oil, 5 mL (depending on thermal
cycler)
*Store on ice
The primer/loading dye mix will turn
purple as the PCR bead dissolves.
If the reagents become splattered
on the wall of the tube, pool them
by pulsing in a microcentrifuge or
by sharply tapping the tube
bottom on the lab bench.
If your thermal cycler does not
have a heated lid: Prior to thermal
cycling, you must add a drop of
mineral oil on top of your PCR
reaction. Be careful not to touch
the dropper tip to the tube or
reaction, or the oil will be
contaminated with your sample.
1. Obtain a PCR tube containing a Ready-To-Go™ PCR Bead. Label with
your assigned number.
2. Use a micropipet with a fresh tip to add 22.5 µL of PTC primer/loading
dye mix to the tube. Allow the bead to dissolve for a minute or so.
3. Use a micropipet with a fresh tip to add 2.5 µL of your cheek cell DNA
(from Part I) directly into the primer/loading dye mix. Insure that no
cheek cell DNA remains in the tip after pipeting.
4. Store your sample on ice until your class is ready to begin thermal cycling.
5. Place your PCR tube, along with other student samples, in a thermal
cycler that has been programmed for 30 cycles of the following
profile. The profile may be linked to a 4°C hold program after the 30
cycles are completed. Complete 35 cycles if you are staining with
CarolinaBLU™.
Denaturing step:
Annealing step:
Extending step:
94°C
64°C
72°C
30 seconds
45 seconds
45 seconds
6. After cycling, store the amplified DNA on ice or at –20°C until you are
ready to continue with Part III.
Copyright © 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.
Using a Single-Nucleotide Polymorphism to Predict Bitter-Tasting Ability
10
III. DIGEST PCR PRODUCTS WITH HaeIII
Reagents (at each student station)
Supplies and Equipment
*PCR product (from Part II), 25 µL
Permanent marker
1.5-mL microcentrifuge tubes
Microcentrifuge tube rack
Micropipet and tips (1–20 µL)
Thermal cycler (or water bath or heat
block)
Container with cracked or crushed ice
Shared Reagent
*Restriction enzyme HaeIII, 10 µL
*Store on ice
The DNA in this tube will not be
digested with the restriction
enzyme HaeIII.
If you used mineral oil during PCR,
pierce your pipet tip through the
mineral oil layer to withdraw the
PCR product in Step 2 and to add
the HaeIII enzyme in Step 3.
1. Label a 1.5-mL tube with your assigned number and with a “U”
(undigested).
2. Use a micropipet with a fresh tip to transfer 10 µL of your PCR
product to the “U” tube. Store this sample on ice until you are ready
to begin Part IV.
3. Use a micropipet with a fresh tip to add 1 µL of restriction enzyme
HaeIII directly into the PCR product remaining in the PCR tube. Label
this tube with a “D” (digested).
4. Mix and pool reagents by pulsing in a microcentrifuge or by sharply
tapping the tube bottom on the lab bench.
Alternatively, you may incubate the
reaction in a 37°C water bath or
heat block. Thirty minutes is the
minimum time needed for
complete digestion. If time
permits, incubate reactions for 1 or
more hours.
5. Place your PCR tube, along with other student samples, in a thermal
cycler that has been programmed for one cycle of the following
profile. The profile may be linked to a 4°C hold program.
Digesting step:
37°C
30 minutes
6. Store your sample on ice or in the freezer until you are ready to begin
Part IV.
IV. ANALYZE PCR PRODUCTS BY GEL ELECTROPHORESIS
Reagents (at each student station)
Supplies and Equipment
*Undigested PCR product
(from Part III), 10 µL
*HaeIII-digested PCR product
(from Part III), 16 µL
Micropipet and tips (1–20 µL)
Microcentrifuge tube rack
Gel electrophoresis chamber
Power supply
Staining trays
Latex gloves
UV transilluminator (for use with
ethidium bromide)
White light transilluminator (for use with
CarolinaBLU™)
Digital or instant camera (optional)
Water bath (60°C)
Container with cracked or crushed ice
Shared Reagents
*pBR322/BstNI marker
2% agarose in 1× TBE, 50 mL
1× TBE, 300 mL
Ethidium bromide (1 µg/mL), 250 mL
or
CarolinaBLU™ Gel and Buffer Stain, 7 mL
CarolinaBLU™ Final Stain, 375 mL
*Store on ice
1. Seal the ends of the gel-casting tray with masking tape, and insert a
well-forming comb.
Copyright © 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.
Using a Single-Nucleotide Polymorphism to Predict Bitter-Tasting Ability
11
Avoid pouring an overly thick gel,
which is more difficult to visualize.
The gel will become cloudy as it
solidifies.
2. Pour 2% agarose solution to a depth that covers about 1/3 the height
of the open teeth of the comb.
Do not add more buffer than
necessary. Too much buffer above
the gel channels electrical current
over the gel, increasing running
time.
4. Place the gel into the electrophoresis chamber, and add enough 1×
TBE buffer to cover the surface of the gel.
100-bp ladder may also be used as
a marker.
6. Use a micropipet with a fresh tip to load 20 µL of pBR322/BstNI size
markers into the far left lane of the gel.
If you used mineral oil during PCR,
pierce your pipet tip through the
mineral oil layer to withdraw the
PCR products. Do not pipet any
mineral oil.
7. Use a micropipet with a fresh tip to add 10 µL of the undigested (U)
and 16 µL of the digested (D) sample/loading dye mixture into
different wells of a 2% agarose gel, according to the diagram below.
Expel any air from the tip before
loading. Be careful not to push the
tip of the pipet through the
bottom of the sample well.
3. Allow the gel to solidify completely. This takes approximately 20
minutes.
5. Carefully remove the comb, and add additional 1× TBE buffer to just
cover and fill in wells—creating a smooth buffer surface.
MARKER
pBR322/
BstNI
STUDENT 1
U
D
STUDENT 2
U
D
STUDENT 3
U
D
8. 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.
Destaining the gel for 5–10
minutes in tap water leeches
unbound ethidium bromide from
the gel, decreasing background
and increasing contrast of the
stained DNA.
9. Stain the gel using ethidium bromide or CarolinaBLU™:
a. For ethidium bromide, stain 10–15 minutes. Decant stain back into
the storage container for reuse, and rinse the gel in tap water. Use
gloves when handling ethidium bromide solution and stained gels or
anything that has ethidium bromide on it. Ethidium bromide is a
known mutagen, and care should be taken when using and disposing
of it.
b. For CarolinaBLU™, follow directions in the Instructor Planning
section.
Transillumination, where the light
source is below the gel, increases
brightness and contrast.
10. View the gel using transillumination, and photograph it using a
digital or instant camera.
Copyright © 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.
Using a Single-Nucleotide Polymorphism to Predict Bitter-Tasting Ability
12
BIOINFORMATICS
For a better understanding of the experiment, do the following bioinformatics
exercises before you analyze your results.
Biological information is encoded in the nucleotide sequence of DNA.
Bioinformatics is the field that identifies biological information in DNA
using computer-based tools. Some bioinformatics algorithms aid the
identification of genes, promoters, and other functional elements of DNA.
Other algorithms help determine the evolutionary relationships between
DNA sequences.
Because of the large number of tools and DNA sequences available on the
Internet, experiments done in silico (in silicon, or on the computer) now
complement experiments done in vitro (in glass, or test tube). This
movement between biochemistry and computation is a key feature of
modern biological research.
http://bioinformatics.dnalc.org/ptc/
animation/index.html
Point your browser to http://bioinformatics.dnalc.org/ptc/ for an onscreen
version of the exercise below guided by David Micklos, founder and Executive
Director of the Dolan DNA Learning Center at Cold Spring Harbor Laboratory.
In Part I, you will use the Basic Local Alignment Search Tool (BLAST) to
identify sequences in biological databases and to make predictions about
the outcome of your experiments. In Part II, you will find and copy the
human PTC taster and non-taster alleles. In Part III, you will discover the
chromosome location of the PTC tasting gene. In Part IV, you will explore
the evolutionary history of the gene.
I.
Use BLAST to Find DNA Sequences in Databases (Electronic PCR)
The following primer set was used in the experiment:
5’-CCTTCGTTTTCTTGGTGAATTTTTGGGATGTAGTGAAGAGGCGG-3’ (Forward Primer)
5'-AGGTTGGCTTGGTTTGCAATCATC-3' (Reverse Primer)
1. Initiate a BLAST search.
a. Open the Internet site of the National Center for Biotechnology
Information (NCBI) www.ncbi.nlm.nih.gov.
b. Click on BLAST in the top speed bar.
c. Click on Nucleotide-nucleotide BLAST (blastn).
d. Enter the sequences of the primers into the Search window. These
are the query sequences.
e. Omit any non-nucleotide characters from the window, because
they will not be recognized by the BLAST algorithm.
f. Click on BLAST! and the query sequences are sent to a server at the
National Center for Biotechnology Information in Bethesda,
Maryland. There, the BLAST algorithm will attempt to match the
Copyright © 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.
forward primer = 5'CCTTCGTTTTCTTGGTGAATTTTTGGGATGTA
GTGAAGAGGCGG-3ʼ
reverse primer =
5'-AGGTTGGCTTGGTTTGCAATCATC-3'
5'-CCTTCGTTTTCTTGGTGAATTTTTGGGATGTAGTGAAGAGGCGG-3ʼ
5'-AGGTTGGCTTGGTTTGCAATCATC-3'
skip
Using a Single-Nucleotide Polymorphism to Predict Bitter-Tasting Ability
17
RESULTS AND DISCUSSION
The following diagram shows how PCR amplification and restriction
digestion identifies the G-C polymorphism in the TAS2R38 gene. The “C”
allele, on the right, is digested by HaeIII and correlates with PTC tasting.
Copyright © 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.
Using a Single-Nucleotide Polymorphism to Predict Bitter-Tasting Ability
18
MARKER
pBR322/
BstNI
tt nontaster
U
D
TT taster
U
D
Tt taster
U
D
MARKER
100 bp
ladder
1857 bp
1058 bp
929 bp
383 bp
221 bp
177 bp
121 bp
44 bp
primer dimer
(if present)
1. Determine your PTC genotype. Observe the photograph of the
stained gel containing your PCR digest and those from other
students. Orient the photograph with the sample wells at the top. Use
the sample gel shown above to help interpret the band(s) in each
lane of the gel.
a. Scan across the photograph to get an impression of what you see
in each lane. You should notice that virtually all student lanes
contain one to three prominent bands.
b. Locate the lane containing the pBR322/BstNI markers on the left
side of the sample gel. Working from the well, locate the bands
corresponding to each restriction fragment: 1857 bp, 1058 bp, 929
bp, 383 bp, and 121 bp. The 1058-bp and 929-bp fragments will be
very close together or may appear as a single large band. The 121bp band may be very faint or not visible. (Alternatively, use a 100-bp
ladder as shown on the right-hand side of the sample gel. These DNA
markers increase in size in 100-bp increments starting with the fastest
migrating band of 100 bp.)
c. Locate the lane containing the undigested PCR product (U). There
should be one prominent band in this lane. Compare the migration
of the undigested PCR product in this lane with that of the 383-bp
and 121-bp bands in the pBR322/BstNI lane. Confirm that the
undigested PCR product corresponds with a size of about 221 bp.
d. To “score” your alleles, compare your digested PCR product (D) with
the uncut control. You will be one of three genotypes:
tt nontaster (homozygous recessive) shows a single band in the
same position as the uncut control.
TT taster (homozygous dominant) shows two bands of 177 bp
and 44 bp. The 177-bp band migrates just ahead of the uncut
control; the 44-bp band may be faint. (Incomplete digestion may
leave a small amount of uncut product at the 221-bp position, but
this band should be clearly fainter than the 177-bp band.)
Copyright © 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.
Using a Single-Nucleotide Polymorphism to Predict Bitter-Tasting Ability
19
Tt taster (heterozygous) shows three bands that represent both
alleles—221 bp, 177 bp, and 44 bp. The 221-bp band must be
stronger than the 177-bp band. (If the 221-bp band is fainter, it is
an incomplete digest of TT.)
e. It is common to see a diffuse (fuzzy) band that runs just ahead of
the 44-bp fragment. This is “primer dimer,” an artifact of the PCR
reaction that results from the primers overlapping one another
and amplifying themselves. The presence of primer dimer, in the
absence of other bands, confirms that the reaction contained all
components necessary for amplification.
f. Additional faint bands at other positions occur when the primers
bind to chromosomal loci other than the PTC gene and give rise to
“nonspecific” amplification products.
2. Determine your PTC phenotype. First, place one strip of control
taste paper in the center of your tongue for several seconds. Note the
taste. Then, remove the control paper, and place one 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, or no taste other than paper?
3. Correlate PTC genotype with phenotype. Record class results in the
table below.
Phenotype
Genotype
Strong taster
Weak taster
Nontaster
TT (homozygous)
Tt
(heterozygous)
tt
(homozygous)
According to your class results, how well does TAS2R38 genotype
predict PTC-tasting phenotype? What does this tell you about
classical dominant/recessive inheritance?
4. How does the HaeIII enzyme discriminate between the C-G
polymorphism in the TAS2R38 gene?
5. The forward primer used in this experiment incorporates part of the
HaeIII recognition site, GGCC. How is this different from the sequence
of the human TAS2R38 gene? What characteristic of the PCR reaction
allows the primer sequence to “override” the natural gene sequence?
Draw a diagram to support your contention.
6. Research the terms synonymous and nonsynonymous mutation.
Which sort of mutation is the G-C polymorphism in theTAS2R38 gene?
By what mechanism does this influence bitter taste perception?
7. Research other mutations in the TAS2R38 gene and how they may
influence bitter taste perception.
Copyright © 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.
20
Using a Single-Nucleotide Polymorphism to Predict Bitter-Tasting Ability
8. The frequency of PTC nontasting is higher than would be expected if
bitter-tasting ability were the only trait upon which natural selection
had acted. In 1939, the geneticist R.A. Fisher suggested that the PTC
gene is under “balancing” selection—meaning that a possible
negative effect of losing this tasting ability is balanced by some
positive effect. Under some circumstances, balancing selection can
produce heterozygote advantage, where heterozygotes are fitter than
homozygous dominant or recessive individuals. What advantage
might this be in the case of PTC?
9. Research how the methods of DNA typing used in this experiment
differ from those used in forensic crime labs. Focus on: a) type(s) of
polymorphism used, b) method for separating alleles, and c) methods
for insuring that samples are not mixed up.
10. What ethical issues are raised by human DNA typing experiments?
Conclusion:
1. How were you able to find and visualize (including knowing exact fragment
lengths) your TAS2R38 genotype? Be sure to include the following in your
explanation: primer, PCR, SNP, RFLP, HaeIII restriction enzyme, gel
electrophoresis, DNA ladder/markers
2. What are haplotypes? What haplotypes are associated with TAS2R38 and
tasters/non-tasters? (see notes and also website: http://www.ncbi.nlm.nih.gov/
entrez/dispomim.cgi?id=607751 Look under Molecular Genetics
3. Describe the structure and function of the TAS2R38 gene product/protein http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=607751 Look under Cloning
4. Describe the locus, # bp, and # exons of the TAS2R38 gene.
http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=607751 Look under Cloning,
Gene Function, Gene Structure
Copyright © 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.
http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=607751!
Gel Analysis
Gel Electrophoresis is a method used to separate biological molecules based upon
molecular charge (polarity), molecular mass, and molecular shape. This is particularly
useful in separating charged biomolecules such as deoxyribonucleic acid, DNA, and
ribonucleic acid, RNA.
The Logger Pro Gel Analysis feature calculates the number of base pairs of various
molecules based on the distance traveled during electrophoresis.
Setup for Gel Analysis
Select Gel Analysis from the Insert menu of the program, and select either Take Photo
or From File. Take Photo is a live method of capturing your gel results using a
ProScope, Logitech, or other digital camera. Choose From File to select a gel
photograph stored in your computer.
Analyzing the Gel Photograph
The following sequence of steps will guide you through analysis of your gel photograph:
1
Click on the Set Origin button and position your cursor to the left of the first well
and click. A yellow origin will appear where vertical and horizontal lines intersect.
Drag the origin up or down to position the horizontal line in the middle of all the
wells. Use the dot on the horizontal line to rotate, if needed.
2
If your photo includes a reference to distance such as a ruler, click on the Set
Scale button. This step is optional. If it is skipped, distances will be reported in
pixels. Use your cursor to draw a line of known distance on the photo. A window
will appear allowing you to input the distance of the line you draw.
3
Click on the Set Standard Ladder button. Click on the center of the first band of
the Standard Ladder in the photo. Enter the number of base pairs in the window
that appears. Click on the next band and repeat for all bands in the standard
ladder.
4
Once the Standard Ladder has been set, click on the Add Lane button and on the
Add Lane option. Click on the center of the first band of the first experimental lane.
The number of base pairs will be calculated and entered in the data table and on
the graph. Click on each of the remaining bands in this lane.
5 To register the bands of the next lane, click on the Add Lane button and on the Add Lane
option that appears. Repeat procedure employed in step 4. This sequence is repeated for
each additional lane being added to your analysis of this gel photograph.
Analysis of your gel is complete.
Note: Logger Pro will automatically name the first experimental lane "Lane 2." If you wish to
change the name, you can do so by double-clicking on the data set name in the data set table
and typing in the new name.
Completed Gel Analysis of plasmid pBR322 digested with three different restriction enzymes
(RE’s). Lanes 6 & 7 exhibit the result of multiple RE’s acting on the plasmid. The central lane
with five bands is the Standard Ladder lane.
The graph depicts the Standard Curve and the large point circles used to establish the curve.
The solid circles represent the standard ladder while the other symbols indicate the different
experimental bands. The graph is scaled to show millimeters (mm) migrated on the horizontal
axis and number of base pairs along the vertical logarithmic axis.