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Ptc_carolina3-10.qxd

Using a Single-Nucleotide Polymorphism to Predict Bitter-Tasting Ability STUDENT LAB INSTRUCTIONS
Mammals are believed to distinguish only five basic tastes: sweet, sour,bitter, salty, and umami (the taste of monosodium glutamate). Tasterecognition is mediated by specialized taste cells that communicate withseveral brain regions through direct connections to sensory neurons.
Taste perception is a two-step process. First, a taste molecule binds to aspecific receptor on the surface of a taste cell. Then, the taste cellgenerates a nervous impulse, which is interpreted by the brain. Forexample, stimulation of "sweet cells" generates a perception of sweetnessin the brain. Recent research has shown that taste sensation ultimately isdetermined by the wiring of a taste cell to the cortex, rather than the typeof molecule bound by a receptor. So, for example, if a bitter taste receptoris expressed on the surface of a "sweet cell," a bitter molecule is perceivedas tasting sweet.
A serendipitous observation at DuPont, in the early 1930s, first showeda genetic basis to taste. Arthur Fox had synthesized somephenylthiocarbamide (PTC), and some of the PTC dust escaped into theair as he was transferring it into a bottle. Lab-mate C.R. Noller complainedthat the dust had a bitter taste, but Fox tasted nothing—even when hedirectly sampled the crystals. Subsequent studies by Albert Blakeslee, atthe Carnegie Department of Genetics (the forerunner of Cold SpringHarbor Laboratory), showed that the inability to taste PTC is a recessivetrait 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 thesurface of taste cells. There are approximately 30 genes for differentbitter 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 positions that vary within the human population—each variable positionis termed a single nucleotide polymorphism (SNP). One specificcombination of the three SNPs, termed a haplotype, correlates moststrongly with tasting ability. Analogous changes in other cell-surface molecules influence the activityof many drugs. For example, SNPs in serotonin transporter and receptorgenes predict adverse responses to anti-depression drugs, includingPROZAC® and Paxil®.
In this experiment, a sample of human cells is obtained by salinemouthwash. DNA is extracted by boiling with Chelex resin, which bindscontaminating metal ions. Polymerase chain reaction (PCR) is then usedto amplify a short region of the TAS2R38 gene. The amplified PCR productis digested with the restriction enzyme HaeIII, whose recognitionsequence includes one of the SNPs. One allele is cut by the enzyme, andone 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, andthen tastes PTC paper. Class results show how well PTC tasting actuallyconforms to classical Mendelian inheritance, and illustrates the modernconcept of pharmacogenetics—where a SNP genotype is used to predictdrug 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.
Copyright 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.
Using a Single-Nucleotide Polymorphism to Predict Bitter-Tasting Ability ISOLATE DNA BY SALINE MOUTHWASH
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 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 Micropipets and tips (10–1000 µL)1.5-mL microcentrifuge tubesMicrocentrifuge tube rackMicrocentrifuge adaptersMicrocentrifugeThermal cycler (or water bath or heat Container with cracked or crushed iceVortexer (optional) Use a permanent marker to label a 1.5-mL tube and paper cup with your assigned number.
Pour saline solution into your mouth, and vigorously rinse your cheek pockets for 30 seconds.
Expel saline solution into the paper cup.
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 Your teacher may instruct you to into your labeled 1.5-mL microcentrifuge tube.
collect a small sample of cells toobserve under a microscope.
Place your sample tube, along with other student samples, in a balanced configuration in a microcentrifuge, and spin for 90 secondsat full speed.
Before pouring off supernatant, Carefully pour off supernatant into the paper cup. Try to remove most check to see that pellet is firmly of the supernatant, but be careful not to disturb the cell pellet at the attached to tube. If pellet is looseor unconsolidated, carefully use bottom of the tube. (The remaining volume will reach approximately micropipet to remove as much the 0.1 mark of a graduated tube.) saline solution as possible. Set a micropipet to 30 µL. Resuspend cells in the remaining saline by pipetting in and out. Work carefully to minimize bubbles.
Food particles will not resuspend.
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 tubewith your assigned number.
Place your PCR tube, along with other student samples, in a thermal The near-boiling temperature lyses cycler that has been programmed for one cycle of the following the cell membrane, releasing DNA profile. The profile may be linked to a 4°C hold program. If you are and other cell contents.
using a 1.5-mL tube, use a heat block or boiling water bath.
Alternatively, you may add the cell suspension to Chelex in a 1.5-mLtube and incubate in a boiling 10. After boiling, vigorously shake the PCR tube for 5 seconds. water bath or heat block.
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 11. Place your tube, along with other student samples, in a balanced tube within sequentially larger configuration in a microcentrifuge, and spin for 90 seconds at full tubes: 0.2 mL within 0.5 mL within speed. If your sample is in a PCR tube, one or two adapters will be 1.5 mL. Remove caps from tubes needed to spin the tube in a microcentrifuge designed for 1.5-mL tubes.
used as adapters.
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 pipettingany 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 Micropipet and tips (1–100 µL) Ready-To-GoTM PCR beads (in 0.2-mL or Microcentrifuge tube rack 0.5-mL PCR tube) Thermal cycler Container with cracked or crushed ice Mineral oil, 5 mL (depending on thermal Obtain a PCR tube containing a Ready-To-Go™ PCR Bead. Label withyour assigned number.
The primer/loading dye mix will turnpurple as the PCR bead dissolves.
Use a micropipet with a fresh tip to add 22.5 µL of PTC primer/loadingdye mix to the tube. Allow the bead to dissolve for a minute or so.
If the reagents become splatteredon the wall of the tube, pool them Use a micropipet with a fresh tip to add 2.5 µL of your cheek cell DNA by pulsing in a microcentrifuge or (from Part I) directly into the primer/loading dye mix. Insure that no by sharply tapping the tubebottom on the lab bench.
cheek cell DNA remains in the tip after pipeting.
If your thermal cycler does not Store your sample on ice until your class is ready to begin thermal cycling.
have a heated lid: Prior to thermalcycling, you must add a drop of Place your PCR tube, along with other student samples, in a thermal mineral oil on top of your PCR cycler that has been programmed to the following profile for 30 reaction. Be careful not to touch cycles if you use ethidium bromide or 35 cycles if you are using the dropper tip to the tube or CarolinaBLU™. The profile may be linked to a 4°C hold program after reaction, or the oil will be cycling is completed.
contaminated with your sample.
Denaturing step: After cycling, store the amplified DNA on ice or at –20°C until you areready 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 III. DIGEST PCR PRODUCTS WITH HaeIII
Reagents (at each student station)
Supplies and Equipment
*PCR product (from Part II), 25 µL Permanent marker1.5-mL microcentrifuge tubes Microcentrifuge tube rack *Restriction enzyme HaeIII, 10 µL Micropipet and tips (1–20 µL)Thermal cycler (or water bath or heat Container with cracked or crushed ice The DNA in this tube will not be 1. Label a 1.5-mL tube with your assigned number and with a "U" digested with the restriction enzyme HaeIII.
2. Use a micropipet with a fresh tip to transfer 10 µL of your PCR If you used mineral oil during PCR, product to the "U" tube. Store this sample on ice until you are ready pierce your pipet tip through the to begin Part IV. mineral oil layer to withdraw thePCR product in Step 2 and to add 3. Use a micropipet with a fresh tip to add 1 µL of restriction enzyme the HaeIII enzyme in Step 3.
HaeIII directly into the PCR product remaining in the PCR tube. Labelthis 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 5. Place your PCR tube, along with other student samples, in a thermal reaction in a 37°C water bath or cycler that has been programmed for one cycle of the following heat block. Thirty minutes is the profile. The profile may be linked to a 4°C hold program.
minimum time needed forcomplete digestion. If time permits, incubate reactions for 1 ormore hours.
6. Store your sample on ice or in the freezer until you are ready to begin IV. ANALYZE PCR PRODUCTS BY GEL ELECTROPHORESIS
Reagents (at each student station)
Supplies and Equipment
*Undigested PCR product Micropipet and tips (1–20 µL) (from Part III), 10 µL Microcentrifuge tube rack *HaeIII-digested PCR product Gel electrophoresis chamber (from Part III), 16 µL Power supplyStaining trays *pBR322/BstNI marker UV transilluminator (for use with 2% agarose in 1× TBE, 50 mL ethidium bromide) White light transilluminator (for use with Ethidium bromide (1 µg/mL), 250 mL Digital or instant camera (optional) CarolinaBLU™ Gel and Buffer Stain, 7 mL Water bath (60°C) CarolinaBLU™ Final Stain, 375 mL Container with cracked or crushed 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 Avoid pouring an overly thick gel, 2. Pour 2% agarose solution to a depth that covers about 1/3 the height which is more difficult to visualize.
of the open teeth of the comb. The gel will become cloudy as itsolidifies. 3. Allow the gel to solidify completely. This takes approximately 20 Do not add more buffer than 4. Place the gel into the electrophoresis chamber, and add enough 1× necessary. Too much buffer above TBE buffer to cover the surface of the gel.
the gel channels electrical currentover the gel, increasing running 5. Carefully remove the comb, and add additional 1× TBE buffer to just cover and fill in wells—creating a smooth buffer surface.
100-bp ladder may also be used as 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, 7. Use a micropipet with a fresh tip to add 10 µL of the undigested (U) pierce your pipet tip through the and 16 µL of the digested (D) sample/loading dye mixture into mineral oil layer to withdraw the different wells of a 2% agarose gel, according to the diagram below.
PCR products. Do not pipet anymineral oil.
Expel any air from the tip beforeloading. Be careful not to push the tip of the pipet through thebottom of the sample well.
8. Run the gel at 130 V for approximately 30 minutes. Adequate separation will have occurred when the cresol red dye front hasmoved at least 50 mm from the wells.
Destaining the gel for 5–10 9. Stain the gel using ethidium bromide or CarolinaBLU™: minutes in tap water leeches a. For ethidium bromide, stain 10–15 minutes. Decant stain back into unbound ethidium bromide fromthe gel, decreasing background the storage container for reuse, and rinse the gel in tap water. Use and increasing contrast of the gloves when handling ethidium bromide solution and stained gels or stained DNA.
anything that has ethidium bromide on it. Ethidium bromide is aknown mutagen, and care should be taken when using and disposingof it.
b. For CarolinaBLU™, follow directions in the Instructor Planning Transillumination, where the light 10. View the gel using transillumination, and photograph it using a source is below the gel, increases digital or instant camera.
brightness and contrast.
Copyright 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.
Using a Single-Nucleotide Polymorphism to Predict Bitter-Tasting Ability For a better understanding of the experiment, do the following bioinformaticsexercises before you analyze your results.
Biological information is encoded in the nucleotide sequence of DNA.
Bioinformatics is the field that identifies biological information in DNAusing computer-based tools. Some bioinformatics algorithms aid theidentification of genes, promoters, and other functional elements of DNA.
Other algorithms help determine the evolutionary relationships betweenDNA sequences.
Because of the large number of tools and DNA sequences available on theInternet, experiments done in silico (in silicon, or on the computer) nowcomplement experiments done in vitro (in glass, or test tube). Thismovement between biochemistry and computation is a key feature ofmodern biological research.
In Part I, you will use the Basic Local Alignment Search Tool (BLAST) toidentify sequences in biological databases and to make predictions aboutthe outcome of your experiments. In Part II, you will find and copy thehuman PTC taster and non-taster alleles. In Part III, you will discover thechromosome location of the PTC tasting gene. In Part IV, you will explorethe evolutionary history of the gene. NOTE: The links in these bioinformatics exercises were correct at the timeof printing. However, links and labels within the NCBI Internet sitechange occasionally. When this occurs, you can find updated exercises athttp://bioinformatics.dnalc.org.
Use BLAST to Find DNA Sequences in Databases (Electronic PCR)
The following primer set was used in the experiment: 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 the link nucleotide BLAST under the heading Basic BLAST.
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. Under Choose Search Set, select the Nucleotide collection (nr/nt) database from the drop-down menu. Copyright 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.
Using a Single-Nucleotide Polymorphism to Predict Bitter-Tasting Ability g. Under Program Selection, optimize for somewhat similar sequences by selecting blastn.
h. 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 theprimer sequences to the millions of DNA sequences stored in itsdatabase. While searching, a page showing the status of yoursearch will be displayed until your results are available. This maytake only a few seconds, or more than a minute if a lot of othersearches are queued at the server.
2. The results of the BLAST search are displayed in three ways as you scroll down the page: a. First, a graphical overview illustrates how significant matches, or hits, align with the query sequence. Matches of differing lengthsare coded by color.
b. This is followed by a list of significant alignments, or hits, with links to Accession information.
c. Next, is a detailed view of each primer sequence (query) aligned to the nucleotide sequence of the search hit (subject). Notice that amatch to the forward primer (nucleotides 1–42), and a match tothe reverse primer (nucleotides 44–68) are within the sameAccession. Also notice that position 43 of the forward primer ismissing. What does this mean? 3. Determine the predicted length of the product that the primer set would amplify in a PCR reaction (in vitro): a. In the list of significant alignments, notice the E-values in the column on the right. The Expectation or E-value is the number ofalignments with the query sequence that would be expected tooccur by chance in the database. The lower the E-value, the higherthe probability that the hit is related to the query. What does the E-value of 6e-12 mean? b. Note the names of any significant alignments that have E-values less than 0.1. Do they make sense? What do they have in common? c. Scroll down to the Alignments section to see exactly where the two primers have landed in a subject sequence.
d. The lowest and highest nucleotide positions in the subject sequence indicate the borders of the amplified sequence.
Subtracting one from the other gives the difference between thetwo coordinates.
e. However, the actual length of the fragment includes both ends, so add 1 nucleotide to the result to determine the exact length of thePCR product amplified by the two primers.
Copyright 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.
Using a Single-Nucleotide Polymorphism to Predict Bitter-Tasting Ability II. Find and Copy the Human (Homo sapiens) PTC Taster and
1. In the list of significant alignments, select the hit containing the human taster allele from among those with the lowest E-values.
2. Click on the Accession link at the left to open the sequence datasheet for this hit.
3. At the top of the report, note basic information about the sequence, including its basepair length, database accession number, source, andreferences.
4. In the middle section of the report, note annotations of gene and regulatory features, with their beginning and ending nucleotide positions(xx . xx). Identify the feature(s) contained between the nucleotidepositions identified by the primers, as determined in 3.d above.
5. The bottom section of the report lists the entire nucleotide sequence of the gene or DNA sequence that contains the PCR product.
Highlight all the nucleotides between the beginning of the forwardprimer and end of reverse primer. Paste this sequence into a textdocument. Then, delete all non-nucleotide characters and spaces. Thisis the amplicon or amplified product.
6. Repeat Steps 1–5 to copy the human non-taster allele. III. Use Map Viewer to Determine the Chromosome Location of the
TAS2R38 Gene
1. Return to the NCBI home page, then click on Map Viewer located in the Hot Spots column on the right.
2. Find Homo sapiens (humans) in the table to the right and click on the "B" icon under the Tools header. If more than one build is displayed,select the one with the highest number, as this will be the mostrecent version.
3. Enter the primer sequences into the search window. Omit any non- nucleotide characters from the window, because they will not berecognized by the BLAST algorithm.
4. Select BLASTN from the drop-down menu under Program and click on Begin Search.
5. Click on View report to retrieve the results.
6. Click on [Human genome view] in the list of Other reports at the top of the page to see the chromosome location of the BLAST hit. On whatchromosome have you landed? 7. Click on the marked chromosome number to move to the TAS2R38 locus.
8. Click on the small blue arrow labeled Genes seq to display genes. The TAS2R38 gene occupies the whole field of the default view, whichdisplays 1/10,000 of the chromosome. Move the zoom out toggle on the Copyright 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.
Using a Single-Nucleotide Polymorphism to Predict Bitter-Tasting Ability left to 1/1000 to see the chromosome region surrounding TAS2R38 andits nearest gene "neighbors." What genes are found on either side ofTAS2R38? How do their structures differ from TAS2R38? Click on theirnames and follow links for more information about them.
9. Click on the blue arrow at the top of the chromosome image to scroll up the chromosome. Look at each of the genes. Scroll up one morescreen, and look at those genes. What do most of these genes have incommon with TAS2R38, and what can you conclude? 10. Zoom out to view 1/100 of the chromosome for a better view of this IV. Use Multiple Sequence Alignment to Explore the Evolution of
TAS2R38 Gene
1. Return to your original BLAST results, or repeat Part I above to obtain a list of significant alignments.
2. Find sequences of the TAS2R38 gene from chimpanzee (Pan troglodytes), bonobo (Pan paniscus), and gorilla. Use only entries listedas "complete cds" (coding sequence). For each, open its Accession link,copy its complete nucleotide sequence from the bottom of thedatasheet, and paste the sequence into a text document.
3. Open the BioServers Internet site at the Dolan DNA Learning Center 4. Enter Sequence Server using the button in the left-hand column. (You can register if you want to save your work for future reference.) 5. Create PTC gene sequences for comparison: a. Click on Create Sequence at the top of the page.
b. Copy one of the TAS2R38 sequences (from Step 2 above), and paste it into the Sequence window. Enter a name for the sequence, andclick OK. Your new sequence will appear in the workspace at thebottom half of the page.
c. Repeat Steps a. and b. for each of the human and primate sequences from Step 2. Also create a sequence for the forwardprimer used in your PCR amplification, and for the amplicon.
6. Compare each of the following sets of sequences: Human PTC taster vs. human PTC non-taster vs. 221 basepairamplicon.
Human PTC taster vs. human PTC non-taster.
Human PCT taster vs. human PTC non-taster vs. chimpanzee vs.
bonobo vs. gorilla.
Forward primer vs. human PTC taster vs. human PTC non-taster.
a. Click on the Check Box in the left-hand column to compare two or more sequences.
Copyright 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.
Using a Single-Nucleotide Polymorphism to Predict Bitter-Tasting Ability b. Click on Compare in the grey bar. (The default operation is a multiple sequence alignment, using the CLUSTAL W algorithm.)The checked sequences are sent to a server at Cold Spring HarborLaboratory, where the CLUSTAL W algorithm will attempt to aligneach nucleotide position.
c. The results will appear in a new window. This may take only a few seconds, or more than a minute if a lot of other searches arequeued at the server.
d. The sequences are displayed in rows of 25 nucleotides. Yellow highlighting denotes mismatches between sequences or regionswhere only one sequence begins or ends before another. e. To view the entire gene, enter 1100 as the number of nucleotides to display per page, then click Redraw.
f. Repeat Steps a–e for each of the four sets of sequences to be g. Human PTC taster vs. human PTC non-taster vs. 221 basepair amplicon. What does the initial stretch of highlighted sequencesmean? Where does the amplicon track along with the two humanalleles? At what position in the gene is the SNP examined in theexperiment, and what is the difference between taster and non-taster alleles? h. Human PTC taster vs. human PTC non-taster. List the nucleotide position(s) and nucleotide differences of any additional SNP(s).
Count triplets of nucleotides from the initial ATG start codon todetermine codon(s) affected by SNP(s). Use a standard geneticcode chart to determine if an amino acid is changed by each SNP.
i. Human PTC taster vs. human PTC non-taster vs. chimpanzee vs.
bonobo vs. gorilla. What is the ancestral (original) state of this geneat nucleotide positions 145, 785, and 886? Are other primatestasters or non-tasters, and what does this suggest about thefunction of bitter taste receptors? What patterns do you notice inSNPs at other locations in the gene? j. Forward primer vs. human PTC taster vs. human PTC non-taster.
Where does the primer bind? What discrepancy do you noticebetween the primer sequence and the TAS2R38 gene sequence?Of what importance is this to the experiment? Copyright 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.
Using a Single-Nucleotide Polymorphism to Predict Bitter-Tasting Ability RESULTS AND DISCUSSION
The following diagram shows how PCR amplification and restrictiondigestion 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 44 bpprimer dimer 1. Determine your PTC genotype. Observe the photograph of the
stained gel containing your PCR digest and those from otherstudents. Orient the photograph with the sample wells at the top. Usethe sample gel shown above to help interpret the band(s) in eachlane 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 lanescontain 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 bandscorresponding to each restriction fragment: 1857 bp, 1058 bp, 929bp, 383 bp, and 121 bp. The 1058-bp and 929-bp fragments will bevery close together or may appear as a single large band. The 121-bp band may be very faint or not visible. (Alternatively, use a 100-bpladder as shown on the right-hand side of the sample gel. These DNAmarkers increase in size in 100-bp increments starting with the fastestmigrating 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 migrationof the undigested PCR product in this lane with that of the 383-bpand 121-bp bands in the pBR322/BstNI lane. Confirm that theundigested 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 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 PCRreaction that results from the primers overlapping one anotherand amplifying themselves. The presence of primer dimer, in theabsence of other bands, confirms that the reaction contained allcomponents 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 thetaste. Then, remove the control paper, and place one strip of PTC tastepaper in the center of your tongue for several seconds. How wouldyou 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.
Weak taster
According to your class results, how well does TAS2R38 genotypepredict PTC-tasting phenotype? What does this tell you aboutclassical 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 sequenceof the human TAS2R38 gene? What characteristic of the PCR reactionallows 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 the TAS2R38 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.
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 selectionhad acted. In 1939, the geneticist R.A. Fisher suggested that the PTCgene is under "balancing" selection—meaning that a possiblenegative effect of losing this tasting ability is balanced by somepositive effect. Under some circumstances, balancing selection canproduce heterozygote advantage, where heterozygotes are fitter thanhomozygous dominant or recessive individuals. What advantagemight 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) ofpolymorphism used, b) method for separating alleles, and c) methodsfor insuring that samples are not mixed up.
10. What ethical issues are raised by human DNA typing experiments? Copyright 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.

Source: http://bioinformatics.dnalc.org/ptc/animation/pdf/ptc_student.pdf