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The Genetics of Taste: Using PTC tasting ability to explore the connections between

genotype and phenotype

Dr. Lynn Miller, Hampshire College and Kathy McCarthy, Amherst Regional High School

Amherst, Mass

Overview: These lessons are designed to transition between understanding inheritance patterns

and the basic structure and function of DNA, to phenotypic expression of traits, accomplished

through transcription/translation of proteins; basically, the connection between genotype and

phenotype. The focus in on the connection between the TAS2R38 gene which is expressed

through the phenotypic ability to taste the bitter compound PTC (phenythiocarbamide). The

activities will explore the inheritance patterns of PTC tasting ability, the location and sequence

of the TAS2R38 gene, the processes of transcription and translation, and will culminate in a lab

that uses current genetic biotechnology to analyze each student's genotype for PTC tasting

ability. [Note: Although the final lab will initially be carried out in Hampshire College’s science

lab, the kit to run this lab is commercially available and can be done in a high school lab setting.]

This series of lessons is designed to guide students, primarily through inquiry-based lessons,

to a better, deeper understanding of the connections between the DNA molecule, genes, the

products of those genes, and the affect of the products on an individual. When students have a

good understanding of these connections, they can then begin to explore what might mediate

those connections. Extensions of these lessons could be in areas such as epigenetics,

transcriptional control, post-transcriptional control, and post-translational control of genes.

Target audience: The first run of these lessons will be used with 2 different Advanced

Placement Biology classes (juniors and seniors). After having experience with the sequence,

modifications will be made for use with introductory College Prep Biology classes.

Misconceptions:

� Genes (themselves) are responsible for the manufacture of proteins

� Dominant genes provide "better" traits

� Mutations only produce diseases

� "Genes" are dominant or recessive (genes - alleles are often interchanged)

Learning Standards (Massachusetts Frameworks):

Biology

3.2 (Describe the basic process of DNA replication and how it relates to the transmission and

conservation of the genetic code). Explain the basic processes of transcription and translation,

and how they result in the expression of genes. Distinguish among the end products of

replication, transcription, and translation.

3.3 Explain how mutations in the DNA sequence of a gene may or may not result in phenotypic

change in an organism.

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Prior knowledge necessary for this sequence of lessons:

a) Inheritance patterns - both Mendelian and non-Mendelian

b) DNA structure and function

c) RNA structures and functions

Major Science Concepts Addressed:

� What is gene expression?

� DNA and RNA structures and functions

� Processes and products of transcription and translation

� Mutations vs. polymorphisms

� Phenotypes and genotypes

� Gene expression

Learning Objectives:

Students will be able to

⋅⋅⋅⋅ distinguish between DNA and RNA

⋅⋅⋅⋅ explain the processes of transcription and translation

⋅⋅⋅⋅ describe the product of transcription and translation

⋅⋅⋅⋅ explain how protein production can be stopped, changed, or modified in different

steps of transcription and translation

⋅⋅⋅⋅ identify the difference between a mutation and a polymorphism in a gene

⋅⋅⋅⋅ interpret electrophoresis gels

⋅⋅⋅⋅ run a PCR-electrophoresis lab

⋅⋅⋅⋅ interpret and apply data to predict phenotype from genotype

Duration: Approximately 5 regular class periods (55 minutes each) and 2 extended lab periods

(90 minutes each)

Materials:

1. Variety of packaged foods that include sour, sweet, bitter, and salty foods

1. Student directions and worksheet for Taste Sensation Lab (Appendix Item A)

2. Predicting PTC inheritance worksheet

3. Transcription-translation activity directions

4. Manipulatives for transcription-translation activity

5. Copies of "Convicted by Juries, Exonerated by Science: Case Studies in the Use of

DNA Evidence to Establish Innocence After Trial", opening comment by Attorney

General Janet Reno (Appendix Item B)

6. Understanding Gel Electrophoresis Lab (Appendix Item C)

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7. Equipment and materials for Gel Electrophoresis Lab

8. Student information sheet for computer resources (Appendix Item D)

9. Using Single-Nucleotide Polymorphism to Predict Bitter Tasting Ability Student

Protocol

10. Lab write-up instructions

Description:

Day 1: Do you have good taste?

[Engage / Explore]

1. Teacher will have a variety of packaged foods on display for students. Students are asked to

describe the different types of taste associated with each food. (Note: packages will NOT be

opened or tasted, they are for discussion purposes only)

2. Inquiry Questions:

1. What different things can you taste?

2. Does everyone taste things to the same degree? Why or why not?

Teacher should guide students to make some connections between taste bud receptors and

genetic instructions that produce the taste buds and taste sensations.

3. Students will complete the Taste Sensation Lab and the analysis questions.

4. Teacher reviews some students answers to analysis questions and presents the following focus

questions: Does your genotype affect your ability to taste a bitter substance? Could you predict

your phenotype based on your genotype?

5. The teacher will provide brief explanation of the inheritance patterns of tasting PTC; students

will complete the Predicting PTC inheritance worksheet.

Day 2: What gives you your sense of taste?

[Explain]

1. Inquiry Questions: (to be discussed separately)

1. What gives you your sense of taste?

2. How do you get from genotype to phenotype?

2. Question #1. This is a good question to talk about as a group, as the teacher will probably

have to guide students toward the ultimate creator of taste – the genes.

3. Question #2: The teacher might precede discussion of this question by having a

representation of the cell (e.g. on board, overhead, ppt) to which students can refer. Teacher,

again, may have to ask guided questions to focus students on important parts of the cell.

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3. Teacher presents a lesson on transcription/translation. [Magnetic white board manipulatives

from Science Kit/Boreal Labs – Protein synthesis chalkboard model - are a great (optional)

resource].

4. (Optional – could be homework) Students will complete on-line transcription-translation

activity : http://learn.genetics.utah.edu/content/begin/dna/transcribe/

5. Students will complete a hands-on transcription-translation activity from

http://serendip.brynmawr.edu/sci_edu/waldron/#trans Scroll down to “From Gene to Protein

activity. Great handouts and instructions.

Day 3 (extended period): How can you determine a person's genotype?

[Explain]

1. Inquiry Questions:

1. Why would you want to know a person’s DNA or genotype?

2. Do we have ways of determining a person’s DNA or genotype? What are they?

2. Discuss inquiry question 1. Encourage discussion about current TV medical dramas or cases

in the news.

3. Hand out copies of article "Convicted by Juries, Exonerated by Science…" in Appendix B for

for students to read. Ask students how DNA is analyzed in order to convict or exonerate

suspects in a crime?

4. Teacher gives a brief explanation of how small pieces of DNA from several sources

are run through gel electrophoresis, then the pattern of DNA fragments are compared for

matches.

3. Students are given the "Student Guide" accompanying the "Understanding Gel

Electrophoresis Lab" {Appendix C}, and asked to review the objectives and background.

4. Teacher gives directions for lab - students complete lab and answer analysis questions.

Day 4: Practicing PCR and Gel Electrophoresis through virtual labs {access to computers

Necessary} {Websites in Appendix D}

1. Give students the student information sheet for computer resources

2. Students will run through the virtual Electrophoresis lab as a review from the previous day.

3. Students will then run through the virtual PCR lab.

4. Students should read through the background information on PTC testing.

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For homework, students should be given a copy of the PTC tasting lab protocol to read and be

prepared for the next day. {Appendix E}

Important note: If you have access to a PCR thermal cycler and gel-electrophoresis equipment,

this lab can be done entirely in a high school classroom

Day 5: Beginning to make connections

Explain/Extend

1. Part 1 of the “Using a single-nucleotide polymorphism to predict PTC tasting ability” will be

conducted in the high school science lab.

2. Students will take samples of their DNA from cheek cells, step I of the lab protocol.

3. Students will amplify their DNA using a PCR thermal cycler. This step will begin during

class; the machine will finish running through the cycles and the DNA will be kept on ice until

beginning the next step.

4. The remaining parts of the lab will be conducted at the Biology lab at Hampshire College.

Day 6: Digesting the DNA

1. Students will take their products from the previous lab and carry out a digest of the DNA

with HaeIII restriction enzymes.

2. While students are waiting for this step to finish, they will use the college computers to

complete a sequencing activity. This activity will help identify the base sequence of the

TAS2R38 gene and compare the human gene with the same gene in other organisms.

Gel Electrophoresis

1. Students will use their restriction digest products and run a Gel electrophoresis

2. The resulting gel will be photographed, and students will make comparisons between

different DNA samples, identifying the types of alleles making up their genotype for tasting

PTC.

3. Based on their identified genotypes, students will then predict if they are a “taster” or a “non-

taster”. Students will be given plain paper and PTC coated paper to test their phenotype.

4. Class will discuss the results and draw conclusions based on looking at genotype and

phenotype. Discussion should include other factors that may affect phenotype.

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Evaluation/Extension

Students will write a research paper that extends their experience with bitter taste:

Which gene(s) influence one other type of taste receptor– sweet, salt, sour, or umami?

Students will report current information about the identity of the gene(s) that affect(s)

these taste receptors and some of the different phenotypes associated with gene(s).

The paper will present a claim about one of the other taste receptors, evidence to support

that claim, and the reasons that the evidence, in fact, support the claim.

{Grading rubric – Appendix F}

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APPENDIX

A: Investigating the Sense of Taste

Materials:

Each group of two students will need

⋅⋅⋅⋅ 4 plastic medicine cups for solutions

⋅⋅⋅⋅ 8 cotton swabs

⋅⋅⋅⋅ 2 plastic cups for water (or a paper plate with celery pieces)

⋅⋅⋅⋅ Paper towels

⋅⋅⋅⋅ 5 ml each of sweet solution, bitter solution, salty solution, and sour solution (see

preparation section for solutions)

⋅⋅⋅⋅ Latex-free gloves

Procedure:

1. One member of the team acts as the experimenter and the other as the subject.

2. Number each of the solution cups 1 – 4, and put 5 ml of each numbered solution in

the appropriate cup.

3. The experimenter will put on a pair of gloves and dip the cotton swab into the first

solution and press out the excess against the medicine cup or paper towel.

4. The subject will stick out his/her tongue and allow the experimenter to place the swab

against the 5 areas of the tongue (see attached diagram).

5. The subject will record if a taste is noticed and what type of taste it is in the data

table.

6. After the first solution is tested, the subject either rinses out his/her mouth or chews a

piece of celery to clean the palate.

7. Discard the swab that was used.

8. Repeat steps 2 through 6 for the other three solutions, one solution at a time.

9. The experimenter and the subject change places and repeat steps 2 – 9.

Observations:

Solution Number Taste? (yes or no) Type of taste noted

1

2

3

4

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Analysis questions:

1. Did your tongue respond to the 4 taste sensations in more than one area? Explain

2. Were your lab partner’s results the same as yours? Explain

3. Where do you think there may be experimental errors in this lab?

4. How do you think this lab could be improved?

5. What other types of taste sensations do you think there might be? How would you test

them?

**Preparation of Solutions:

⋅⋅⋅⋅ Sweet: 2 tsp. sugar + 250 ml water

⋅⋅⋅⋅ Sour: 30 ml vinegar + 30 ml water

⋅⋅⋅⋅ Salty: 2 tsp. salt + 250 ml water

⋅⋅⋅⋅ Bitter: 2 aspirin* + 250 ml water

* you may want to substitute a different bitter solution

[NOTE: You should check to see if a parental permission slip is necessary for students to taste

materials in the lab. IF you use aspirin, you definitely need to have a parental permission slip.

You must be aware of student allergies in your classroom]

B: Electronic source for article: “Convicted by Juries, Exonerated by Science: Case

Studies in the Use of DNA Evidence

http://www.ncjrs.gov/txtfiles/dnaevid.txt (NOTE: This is a 124 page article – I used the first

2 pages – the introduction by Janet Reno)

C: Understanding Agarose Gel Electrophoresis Lab: Prepackaged kit from Neoscience

http://www.neoscience.com : Item #20-3303

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D: Student Computer Information Services

Virtual Gel Electrophoresis:

http://learn.genetics.utah.edu/units/biotech/gel/

Virtual PCR Lab:

http://learn.genetics.utah.edu/units/biotech/pcr

PTC tasting background information:

http://learn.genetics.utah.edu/units/basics/ptc

NPR story on the background of different tastes: “Sweet, Sour, Salty, Bitter, and….Umami

http://www.npr.org/templates/story/story.php?storyid=15819485

Article about geographical patterns associated with taste: “The geography of taste”

http://www.tastescience.com/abouttaste4.html

E. PTC tasting lab protocol

A kit with supplies and a wonderful introduction and directions is available from Carolina

Biological Supply { Using a Single Nucleotide Polymorphism (SNP) to Predict Bitter Tasting Ability} This lab was developed through the Dolan DNA Learning Center at

Cold Spring Harbor Laboratory.

The following is a modification of that lab for our particular situation and equipment:

PTC Tasting Ability Student Protocol 4/5/09 MRN (modified protocol from Carolina Kit)

Isolating Your DNA by Saline Mouthwash

Each student will have a station with a 15mL centrifuge tube containing 0.9% Saline solution

(NaCl);

a permanent marker, paper cup; micropipets and tips, 1.5mL microcentrifuge (µfuge) tubes and a

µfube tube with 100µL of Chelex.

1) Use the permanent marker to label two 1.5mL µfuge tubes with your name or assigned

number;

2) Pour the saline solution into you mouth and swish vigorously for 30 seconds; you may scrape

your cheeks with your teeth to get more cheek cells;

3) Expel saline into paper cup;

4) Swirl the cup gently; using a 1000µL pipetter and tip transfer 1000µL of your wash into your

labeled µfuge tube;

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5) Place your µfuge tube in the µcentrifuge; tubes must be balanced; spin full speed 90 seconds;

6) Pour off the supernatant fluid from your tube into the paper cup; be careful not to disturb the

cell pellet at the bottom of the tube; [There will be some left over fluid in your tube.]

7) Set a µpipet to 30µL; resuspend the cells by pipetting gently in and out;

8) Add your resuspended pellet from tube into a µfuge tube containging 100µl of Chelex

suspension; take to boiling water bath;

9) Boil in boiling water bath for 10min;

10) After boiling, shake tube vigorously for 5 seconds;

11) Spin for 90 sec in mini µfuge;

12) Pipet 30µL of the clear supernatant fluid into a labeled 1.5mL tube- this is your DNA sample

to set

up PCR reactions- save in ice or @ -20C .

II. Amplify DNA by PCR

Obtain a PCR tube containing a Ready-To-Go PCR Bead. Label with assigned number.

1) Use a fresh tip to add 22.5 µL of PTC primer/loading dye mix to the tube. Allow bead to

dissolve for 1 to 2 minutes.

2)With a fresh tip add 2.5 µL of your cheek cell DNA directly into the primer/loading dye

mix. Make sure no cheek cell DNA remains on the tip after transfer.

3) Store sample on ice until class is ready to begin thermal cycling.

4) Place PCR tube in programmed thermal cycler .

Method: taste-64c

32 cycles

Denaturing step: 94C 30 seconds

Annealing step: 64C 45 seconds

Extending step: 72C 45 seconds

Final hold 4C forever

5)After cycling, store tube on ice until class is ready to continue.

*After loading the cycler, we will discuss the rest of the experiment and practice loading,

running and analyzing gels with a separate kit. The remaining steps will be preformed after

students leave, and results will be emailed at the end of the day.

III. Digest PCR products with HaeIII (This will be done after class leaves)

1. Label a 1.5mL tube with assigned number and with a “U” (undigested)

2. Use a fresh tip to transfer 10µL of PCR product into tube “U”. Store on ice until next

step.

3. With a fresh tip add 1µL of restriction enzyme directly into the PCR product remaining in

tube. Label this tube “D” (digested).

4. Mix and pool reagents by pulsing in microcentrifuge.

5. Place PCR tube in thermal cycler programmed for 37C for 60 minutes.

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6. Store on ice or in freezer until next step.

IV. Analyze PCR products by Gel Electrophoresis

The agar used in this part of the experiment contains Ethidium Bromide. Use gloves and

reasonable care when handling.

1) Obtain small gel form with an 8 well comb.

2) Level the device and pour 40mL 2% agar to a height about 1/3 up the comb.

3) Allow gel to harden for at least 20 minutes.

4) Remove the comb carefully and place the gel into the electrophoresis chamber.

5) Add 1xTBE to both sides of the chamber and stop filling after the gel surface is covered and

wells are submerged.

6) Use a µpipet with a fresh tip to load 20µL of pBR322/BstNI size markers into the far left

lane of the gel.

7) Using a fresh tip for each, add 10µL of the undigested (U) sample into one well and 16µL

of the digested (D) sample into another.

1 2 3 4 5 6 7 8

Size

marker

Student1

U

Student1

D

Student2

U

Student2

D

Student3

U

Student3

D

Empty/

control

8) Run the gel at 160 V for approximately 30 minutes.

9) View the gel in the Gel Doc apparatus and photograph it.

* Loaded samples may be of smaller volumes, to accommodate running a second type of gel in

addition to the typical agar one.

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Appendix F:

Project Report Rubric Genetic

basis of taste

Component LEVEL

2 1 0

CLAIM

A conclusion that

answers the

original question

Makes an accurate

and complete claim

about the

relationship

between gene and

type of taste

Makes an accurate

but incomplete

claim

Does not make a

claim, or makes an

inaccurate claim

EVIDENCE

Scientific data

that supports the

claim. The data

needs to be

appropriate and

sufficient to

support the claim.

Provides

appropriate and

sufficient

evidence to

support the claim.

Evidence is from

reliable source(s).

Provides

appropriate, but

insufficient

evidence to

support claim or

has sufficient

evidence, but is

unreliable.

Does not provide

evidence, or only

provides

inappropriate or

unreliable evidence.

REASONING

A justification

that links the

claim and

evidence. It

explains why the

data can be used

as evidence.

Provides reasoning

that links evidence

to claim. Includes

appropriate and

sufficient

scientific

principles.

Provides reasoning

that links claim

and evidence, but

repeats some

evidence and/or

includes

insufficient

evidence.

Does not provide

reasoning or only

provides reasoning

that does not link

evidence to claim.

(Extra credit)

REBUTTAL

Recognizes and

describes

alternative

explanations,

provides counter

evidence, and

reasoning for why

alternative

explanation is not

appropriate

Recognizes

alternative

explanations and

provides

appropriate and

sufficient counter

evidence and

reasoning.

Recognizes

alternative

explanations and

provides

appropriate, but

insufficient

counter evidence

and/or reasoning.

Recognizes

alternative

explanations, but

does not provide

evidence or

reasoning in

rebuttal.

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Kathy McCarthy and Lynn Miller Reflection and follow up to project

Before getting into the details of our project, I would like to say that this collaboration

worked extremely well and the results were exciting and encouraging. I felt we were both

prepared, organized, and had a clear idea of what we wanted to accomplish. The focus, and most

important part of this project, was the students. To that end, the high school students had a

unique lab experience, an opportunity to work with college students, and chance to apply

classroom knowledge to real-world problems.

The most difficult piece of the puzzle was getting our schedules to mesh. Hampshire

College runs on a semester system, while Amherst Regional High School is on a trimester

system. That means the AP Biology course, for which the project was originally designed, meets

for only 2 trimesters, or until early March. The genetics unit happened to fall during Hampshire

college’s holiday break. By the time students were back and we could start scheduling, the AP

class was nearing the end and hard pressed to find any extra time to dedicate to this lab.

We were very persistent, however, and were finally able to find a compatible date in late April.

Our second obstacle was how to choose students to participate. The AP class was over,

and I was teaching 3 College Prep level (our lowest level) Biology classes. There were also

concerns about the number of students Hampshire College could accommodate at one time. I

decided to choose a few of my top students in each of my classes, plus a couple students from

another AP Biology class to participate. This turned out to be one of the best parts of the project,

as normally overlooked students were recognized for their effort, and we could see how the lab

could be adapted to other academic levels. Several of these students were not necessarily

planning on a future in a science-related field, but they were able to see another side of science.

At the very least, they will be better-informed adults, and perhaps it may have ignited a spark of

interest in genetics.

Our original plan was to complete part of the lab at the high school and part at Hampshire

College. Due to lack of time and equipment, we adapted the lab for using only the Hampshire

College facilities. Some of the time consuming parts of the lab, such as the digests, were done

by Hampshire College student lab assistants after the high school students left, and results were

e-mailed. This worked fine, as students were able to practice techniques, set the steps, and get

their results. We used their results in discussions about genotype and phenotype.

We are planning to continue this collaboration into the future. Certainly there are details

to work out, especially in terms of timing, but it was a great success. The most important part of

the collaboration was the excitement and learning opportunities for both the high school and

college students.

PTC experiment performed 4/17/09

ARHS student Taste-Test Genotype (see gels)

1 Surya Murty No Non-taster

2 Laura Woodbridge Yes taster

3 Amanda Glaszcz No Non-taster

4 Olivia Holcomb Yes Homozygous taster

6 Otis Rowell Yes taster

7 Ezra Ward Yes taster

8 Emma Ayers Yes SORRY pcr did not work

11 Tristan Kovacs No taster

13 Laura Bustamante Yes taster

14 Spencer Kaye Yes taster

Many of the bands are very faint, and the DNA samples are at different concentrations, the photographs

of the gels are the best for all. Most of the genotyping that seems unclear was actually visible when the

contrast etc… was manipulated.

Crime simulation (gel run at 150V for 60 minutes)

The evidence “collected” was digested with several standard enzymes, these enzymes produce a DNA

finger print unique enough to connect or eliminate a suspect.

Two suspects – X and –Y had DNA collected and digested with the same enzymes. The digested

products were run on a gel by Kathy MacCarthy’s students. Loaded as follows:

1 2 3 4 5 6 7 8

X-1 X-2 Y-1 Y-2 E-1 E-2 Size

marker

Empty

Based on the gel:

It appears that Suspect X can be eliminated. Suspect

Y may be guilty.

PTC–PCR/ Digest results

Two gels loaded as follows:

1 2 3 4 5 6 7 8 9 10 11 12

D-1 U-1 D-2 U-2 D-3 U-3 D-4 U-4 D-6 U-6 D-11 U-11

D-Means digested with HaeIII restriction enzyme

U-Means undigested portion of DNA to use a control

1 2 3 4 5 6 7 8

D-7 U-7 D-8 U-8 D-13 U-13 D-14 U-14