Lesson 3: you taste PTC? taste PTC, where the number of heterozygotes + B homozygotes that taste PTC...

K. McCarthy, L. Miller, ASHG GENA 2008 Cohort

Learning Cycle Stage(s): Elaborate/Evaluate

Adapted from: The Gene cs of Taste Kathy McCarthy, Amherst Regional High School, MA Lynn Miller, Hampshire College, MA

ASHG Concept(s) Addressed: Transmission/pa erns of inheritance #3, #5

Time Required: 325‐395 minutes

Elaborate 1) In this lab, students will use DNA extrac on, PCR, restric on enzyme diges on, and agarose gel

electrophoresis to determine their genotype for the TAS2R38 bi er taste receptor, the gene impli‐cated in the ability to taste PTC or not. The kit they will follow, Using a Single Nucleo de Polymor‐phism (SNP) to Predict Bi er Tas ng Ability Kit, was developed by the Dolan DNA Learning Center and is available through Carolina Biological (h p://www.carolina.com/human‐gene cs/using‐a‐single‐nucleo de‐polymorphism‐snp‐to‐predict‐bi er‐tas ng‐ability‐kit/FAM_211376.pr?catId=&mCat=&sCat=&ssCat=&ques on=Using+a+Single+Nucleo de+Polymorphism+(SNP)+to+Predict+Bi er+Tas ng+Ability+Kit).

2) Students should read over pages 5‐11 of the Student Lab Instruc ons (Appendix I) and write down any ques ons they have.

3) In the Introduc on (pg. 5‐6) of the Student Lab Instruc ons, the author states “One specific combi‐na on of the three [TAS2R38] SNPs, termed a haplotype, correlated most strongly with tas ng abil‐ity .” Discuss this statement with the class, asking students what it means for a genotype (either a haplotype as described or a single SNP like the one they will test) to be correlated with a pheno‐type. Does correla on between a genotype and a phenotype imply causa on? Why or why not? What cons tutes a meaningful correla on (for example, even correla ons between causa ve gen‐otypes and their phenotypes are not always 100%, as in the case of polygenic phenotypes or in‐complete penetrance)?

4) Ask student pairs to determine how they would test the rela onship between TAS2R38 genotype and PTC phenotype among their fellow students. What data would they collect? How would they analyze those data to determine if genotype and phenotype are related in this case? Given the data they intend to collect, task students with coming up with a descrip ve sta s c for the data that will be meaningful in its interpreta on (for example, 75% of people with at least one B allele can taste PTC, where the number of heterozygotes + B homozygotes that taste PTC strongly and weakly is divided by the number of total heterozygotes + B homozygotes). More advanced stats that can be used to evaluate this correla on include chi square with a 3x3

con ngency table, or the related Cramer’s Phi Coefficient. 5) Before beginning the lab, discuss any student ques ons arising from the Student Lab Instruc ons,

especially those that concern the technical details of the protocol. To make sure students under‐stand why they are performing each step, we propose some ques ons before each phase of the experiment. Students should discuss with each other as necessary, and/or as a class, and record their answers.

6) This kit breaks down into the following chunks of lab ac vity: a. Isolate DNA by saline mouthwash.

Before students begin, ask, What is the first step you need to complete to determine your genotype? What physical processes does this entail and how do they take advantage of the

HW

10 min

15 min

5 min

30 min

The Gene cist‐Educator Network of Alliances Project ● NSF EHR#0634296 ● www.ashg.org/educa on/gena.shtml

Lesson 3: Can you taste PTC?

http://www.carolina.com/human-genetics/using-a-single-nucleotide-polymorphism-snp-to-predict-bitter-tasting-ability-kit/FAM_211376.pr?catId=&mCat=&sCat=&ssCat=&question=Using+a+Single+Nucleotide+Polymorphism+(SNP)+to+Predict+Bitter+Tasting+Ability+Kit�

















structure of the cells and the chemical proper es of DNA? b. Amplify DNA by PCR.

Before students begin, ask, What is the purpose of this step? Why can’t you go straight from isola ng DNA to cu ng it with your restric on enzyme? What ingredients and/or steps ensure that you will amplify the piece of DNA you intend to amplify during a PCR re‐ac on?

This is a great me to introduce students who do not have experience with gel electropho‐resis to the delicate task of loading a gel. Submerge a prac ce gel (does not need to con‐tain EtBr) in buffer and allow students to take turns loading 5‐10 µL of loading dye into the wells. (6X dye can be purchased cheaply from many laboratory supply companies.) Stu‐dents should prac ce un l they feel comfortable with their ability to load wells without piercing them or pipe ng the sample into the buffer.

c. Digest PCR products with HaeIII. Before students begin, ask, What is the purpose of this step? What chemical or physical

factors determine where restric on enzymes will cut a piece of DNA? To save me during the next sec on, gels can be poured while the digests are incuba ng. If

they won’t be run un l the next day, wrap them in plas c wrap so they don’t dry out and refrigerate.

d. Analyze diges on products by gel electrophoresis. Before students begin, ask, What is the purpose of this step? Why are we running both the

undigested and the digested PCR product out on the gel? What is the purpose of the size markers?

While the gels are running, discuss experimenter bias with the class. How was bias avoided in the taste experiment in lesson 1? How can students use those strategies to ensure that they get the most reliable PTC tas ng phenotype data? As a class, students should design a blinded taste test for PTC and student pairs should determine their PTC tas ng pheno‐types using that experimental design (strongly bi er, weakly bi er, no taste).

e. If your school does not have a thermacycler or gel electrophoresis equipment, consider con‐tac ng a local college/university or biotech company. Labs may have equipment they may be willing to donate or lend.

f. Throughout the lab, students should take notes on the procedures they are doing, the goal of each step, and any ques ons they may have.

7) A er the gels have been photographed, have students work in pairs to interpret their gel bands and determine the combina on of alleles they possess. A er students have determined their gen‐otype, collect class data on genotype and phenotype using the table in Appendix II.

As noted in the kit’s instruc ons, presence of cut and intact PCR product should only be interpreted as a heterozygous genotype if the uncut (221 bp) band is brighter than the smaller bands. If the uncut band is fainter, it is more likely to be incompletely digested PCR product from the taster allele.

8) Allow each pair of students to analyze the class data using their descrip ve sta s c. Discuss results as a class. What are the advantages and/or caveats of each descrip on?

Evaluate Based on their notes, students should write a full lab report including the following elements. Por ons of this report can be worked on during lab down me.

a. the overall ques on being inves gated in this lab

15 min for prep; 90‐120 min for cycling

15 min for prep; 30 min for digest

15‐45 min for prep; 30 min to run, 20 min to

stain, and 20 min to photo‐

graph gels

20 min

10‐20 min

HW



b. background on how the sense of taste works c. hypothesized RFLP results for each of the possible TAS2R38 genotypes d. a summary of the methods used to determine genotype and phenotype that includes the pur‐

pose of each step in the genotyping protocol and a conceptual explana on of how that step works (not the detailed procedural steps)

e. a descrip on of their personal genotype/phenotype data as well as the class’s genotype/phenotype data

f. an analysis/interpreta on of the data that relates back to the ques on being inves gated g. a discussion of the data in light of what they know about Mendelian dominant and recessive

modes of inheritance (Do the class data suggest Mendelian inheritance of PTC tas ng ability? If so, which TAS2R38 allele and associated phenotype appears to be dominant and which ap‐pears to be recessive?)

h. iden fica on of data that do not agree with the general trend of the class data and discussion of at least one experimental reason those data may not agree and one biological reason those data might not agree. How would they determine whether the data are most likely the result of experimental error or the result of true biological difference?


klontok

Typewritten Text

klontok

Typewritten Text

5Using a Single-Nucleotide Polymorphism to Predict Bitter-Tasting Ability

Copyright © 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.

STUDENT LAB INSTRUCTIONS

INTRODUCTION

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.

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

Albert Blakeslee using a votingmachine to tabulate results of

taste tests at the AAASConvention, 1938. (Courtesy Cold

Spring Harbor LaboratoryResearch Archives)

klontok

Typewritten Text

Appendix I

klontok

Typewritten Text

Learning CenterDNA KITS

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). PositionalCloning of the Human Quantitative Trait Locus Underlying Taste Sensitivity toPhenylthiocarbamide. 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.

Using a Single-Nucleotide Polymorphism to Predict Bitter-Tasting Ability


6


– +

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




8

Your teacher may instruct you tocollect a small sample of cells toobserve under a microscope.

Before pouring off supernatant,check to see that pellet is firmlyattached to tube. If pellet is looseor unconsolidated, carefully usemicropipet to remove as muchsaline solution as possible.

METHODS

I. ISOLATE DNA BY SALINE MOUTHWASH

1. Use a permanent marker to label a 1.5-mL tube and paper cup withyour assigned number.

2. Pour saline solution into your mouth, and vigorously rinse your cheekpockets for 30 seconds.

3. Expel saline solution into the paper cup.

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 solutioninto your labeled 1.5-mL microcentrifuge tube.

5. Place your sample tube, along with other student samples, in abalanced configuration in a microcentrifuge, and spin for 90 secondsat full speed.

6. Carefully pour off supernatant into the paper cup. Try to remove mostof the supernatant, but be careful not to disturb the cell pellet at thebottom of the tube. (The remaining volume will reach approximatelythe 0.1 mark of a graduated tube.)

7. Set a micropipet to 30 µL. Resuspend cells in the remaining saline bypipetting in and out. Work carefully to minimize bubbles.

8. Withdraw 30 µL of cell suspension, and add it to a PCR tubecontaining 100 µL of Chelex®. Label the cap and side of the tubewith your assigned number.

9. Place your PCR tube, along with other student samples, in a thermalcycler that has been programmed for one cycle of the followingprofile. The profile may be linked to a 4°C hold program. If you areusing 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.

Reagents (at each student station)

0.9% saline solution, 10 mL10% Chelex®, 100 µL (in 0.2- or 0.5-mL PCR

tube)

Supplies and Equipment

Permanent markerPaper cupMicropipets and tips (10–1000 µL)1.5-mL microcentrifuge tubesMicrocentrifuge tube rackMicrocentrifuge adaptersMicrocentrifugeThermal cycler (or water bath or heat

block)Container with cracked or crushed iceVortexer (optional)

Food particles will not resuspend.

The near-boiling temperature lysesthe cell membrane, releasing DNAand other cell contents.

Alternatively, you may add the cellsuspension to Chelex in a 1.5-mLtube and incubate in a boilingwater bath or heat block.



11. Place your tube, along with other student samples, in a balancedconfiguration in a microcentrifuge, and spin for 90 seconds at fullspeed. If your sample is in a PCR tube, one or two adapters will beneeded 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 clearsupernatant 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. Thissample 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 continuewith Part II.

II. AMPLIFY DNA BY PCR

1. Obtain a PCR tube containing a Ready-To-Go™ PCR Bead. Label withyour assigned number.

2. 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.

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 nocheek 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 thermalcycler that has been programmed to the following profile for 30cycles if you use ethidium bromide or 35 cycles if you are usingCarolinaBLU™. The profile may be linked to a 4°C hold program aftercycling is completed.

Denaturing step: 94°C 30 seconds Annealing step: 64°C 45 secondsExtending step: 72°C 45 seconds

6. After cycling, store the amplified DNA on ice or at –20°C until you areready to continue with Part III.

To use adapters, “nest” the sampletube within sequentially largertubes: 0.2 mL within 0.5 mL within1.5 mL. Remove caps from tubesused as adapters.

The primer/loading dye mix will turnpurple as the PCR bead dissolves.


*Cheek cell DNA, 2.5 µL (from Part I)*PTC primer/loading dye mix, 22.5 µLReady-To-GoTM PCR beads (in 0.2-mL or

0.5-mL PCR tube)

Shared Reagent

Mineral oil, 5 mL (depending on thermalcycler)

*Store on ice


Permanent markerMicropipet and tips (1–100 µL)Microcentrifuge tube rackThermal cycler Container with cracked or crushed ice

If the reagents become splatteredon the wall of the tube, pool themby pulsing in a microcentrifuge orby sharply tapping the tubebottom on the lab bench.

If your thermal cycler does nothave a heated lid: Prior to thermalcycling, you must add a drop ofmineral oil on top of your PCRreaction. Be careful not to touchthe dropper tip to the tube orreaction, or the oil will becontaminated with your sample.




10

III. DIGEST PCR PRODUCTS WITH HaeIII

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 PCRproduct to the “U” tube. Store this sample on ice until you are readyto begin Part IV.

3. Use a micropipet with a fresh tip to add 1 µL of restriction enzymeHaeIII 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 sharplytapping the tube bottom on the lab bench.

5. Place your PCR tube, along with other student samples, in a thermalcycler that has been programmed for one cycle of the followingprofile. 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 beginPart IV.

IV. ANALYZE PCR PRODUCTS BY GEL ELECTROPHORESIS

1. Seal the ends of the gel-casting tray with masking tape, and insert awell-forming comb.

The DNA in this tube will not bedigested with the restrictionenzyme HaeIII.

If you used mineral oil during PCR,pierce your pipet tip through themineral oil layer to withdraw thePCR product in Step 2 and to addthe HaeIII enzyme in Step 3.


*PCR product (from Part II), 25 µL

Shared Reagent

*Restriction enzyme HaeIII, 10 µL

*Store on ice


Permanent marker1.5-mL microcentrifuge tubesMicrocentrifuge tube rack Micropipet and tips (1–20 µL)Thermal cycler (or water bath or heat

block)Container with cracked or crushed ice


*Undigested PCR product (from Part III), 10 µL

*HaeIII-digested PCR product (from Part III), 16 µL

Shared Reagents

*pBR322/BstNI marker2% agarose in 1× TBE, 50 mL1× TBE, 300 mL Ethidium bromide (1 µg/mL), 250 mL orCarolinaBLU™ Gel and Buffer Stain, 7 mLCarolinaBLU™ Final Stain, 375 mL

*Store on ice


Micropipet and tips (1–20 µL)Microcentrifuge tube rackGel electrophoresis chamberPower supplyStaining traysLatex glovesUV transilluminator (for use withethidium bromide)

White light transilluminator (for use withCarolinaBLU™)

Digital or instant camera (optional) Water bath (60°C)Container with cracked or crushed ice

Alternatively, you may incubate thereaction in a 37°C water bath orheat block. Thirty minutes is theminimum time needed forcomplete digestion. If timepermits, incubate reactions for 1 ormore hours.

2. Pour 2% agarose solution to a depth that covers about 1/3 the heightof the open teeth of the comb.

3. Allow the gel to solidify completely. This takes approximately 20minutes.

4. Place the gel into the electrophoresis chamber, and add enough 1×TBE buffer to cover the surface of the gel.

5. Carefully remove the comb, and add additional 1× TBE buffer to justcover and fill in wells—creating a smooth buffer surface.

6. Use a micropipet with a fresh tip to load 20 µL of pBR322/BstNI sizemarkers into the far left lane of the gel.

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 intodifferent wells of a 2% agarose gel, according to the diagram below.

8. Run the gel at 130 V for approximately 30 minutes. Adequateseparation will have occurred when the cresol red dye front hasmoved at least 50 mm from the wells.

9. Stain the gel using ethidium bromide or CarolinaBLU™:

a. For ethidium bromide, stain 10–15 minutes. Decant stain back intothe storage container for reuse, and rinse the gel in tap water. Usegloves when handling ethidium bromide solution and stained gels oranything 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 Planningsection.

10. View the gel using transillumination, and photograph it using adigital or instant camera.



Transillumination, where the lightsource is below the gel, increasesbrightness and contrast.

Destaining the gel for 5–10minutes in tap water leechesunbound ethidium bromide fromthe gel, decreasing backgroundand increasing contrast of thestained DNA.

Avoid pouring an overly thick gel,which is more difficult to visualize.The gel will become cloudy as itsolidifies.

Do not add more buffer thannecessary. Too much buffer abovethe gel channels electrical currentover the gel, increasing runningtime.

100-bp ladder may also be used asa marker.

If you used mineral oil during PCR,pierce your pipet tip through themineral oil layer to withdraw thePCR products. Do not pipet anymineral oil.

Expel any air from the tip beforeloading. Be careful not to push thetip of the pipet through thebottom of the sample well.

MARKER STUDENT 1 STUDENT 2 STUDENT 3pBR322/ U D U D U D

BstNI


Appendix II


Genotype Strong Taster Weak Taster Non‐taster

TATA

TATB

TBTB

Phenotype

Lesson 3: you taste PTC? taste PTC, where the number of heterozygotes + B homozygotes that taste PTC...

Documents

Transcript of Lesson 3: you taste PTC? taste PTC, where the number of heterozygotes + B homozygotes that taste PTC...