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Testing Your Taste: PTC (Taster) Gene Phenotype & Genotype Analysis

For Smith College Interterm Science Teaching Internship at the Northampton High School.
by

Sarah Banks Winokur

on 3 February 2013

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Transcript of Testing Your Taste: PTC (Taster) Gene Phenotype & Genotype Analysis

PTC (Taster) Gene Phenotype & Genotype Analysis Testing Your Taste Overview
of the next few days... With your Smith College Interns:
Arcadia,
Sarah, and Charlene! PTC Taster
Phenotype Day 1: Day 2: Extract DNA
from Cheek Cells Taster Gene PCR PCR Product Digestion
with Restriction Enzyme Special Activities! Gel Electrophoresis of
Digested PCR Product PTC Taster
Genotype Analysis! Day 1 1. PTC Papers
-Record if you are a Taster or Nontaster 2. Materials and Pipetting Demonstration 3. Experiment (Steps 1-3) 4. Taste Discussion PTC (phenylthiocarbamide)

Tastes bitter

Some people's PTC genes code for a receptor protein that binds to PTC

Binding sends a “bitter” signal to brain

Other people have a few base pair differences in their PTC gene that causes them to have receptors that do not bind to PTC

PTC does not taste bitter to these people because they do not have surface proteins that bind to PTC and send the “bitter” signal PTC 5 basic tastes: Taste Taste buds: clusters of taste receptor cells within papillae
5,000 taste buds
Up to 150 taste receptors per taste bud Taste Receptors Foods containing acids taste sour, e.g. citrus fruits… Sour Taste Receptor proteins in cell membranes (in the taste buds on the tongue) bind to specific molecules in food
Sends a signal which causes a series of chemical reactions in the cell
Signal causes release of chemicals which stimulate the sensory nerve which sends the signal to the brain Taste Signal Pathway Foods containing amino acids, such as glutamate, taste savory e.g. cheese, bacon… Umami Taste Foods containing some plant compounds, such as alkaloids, taste bitter, e.g. some vegetables, coffee… Bitter Taste Foods containing sodium chloride or other salts taste salty, e.g. chips, pretzels, ketchup… Salty Taste Foods containing glucose and other simple sugars taste sweet, e.g. fruit, candy… Sweet Taste 5. Genetics Discussion Sweet Sour Salty Bitter Umami 6. Experiment (Steps 5-7) 7. PCR Introduction Day 2 A Punnett Square shows the expected results of a genetic cross
For example: TT x tt, Tt x tt, Tt x Tt
-Homozygous
-Heterozygous
-Genotype
-Phenotype Punnett Squares You’ve noticed that some people are tasters and others are non-tasters… why is this?

The ability to taste PTC is controlled
by genetic differences in the PTC gene The Building Blocks of Genetics
Phenotype: The physical appearance of an organism Review of Vocabulary
One allele can mask the other
Ex: Aa


A parent can pass on only one allele per trait
Ex: Parent is Aa… Pass on A or a


Traits are inherited independently of each other Mendel’s 3 Laws DNA = Deoxyribonucleic acid

DNA is genetic material
1. Replicate
2. Carry Information
3. Change DNA Mendelian Genetics What are the mechanisms by which traits are inherited? What is DNA made of? Nucleotides!

-Sugar: 2 Deoxyribose


-Phosphate:

-Base:
[A] = [T]
[C] = [G] DNA The Central Dogma of genetics is the flow of genetic information within an organism (DNA to RNA to Protein)

The PTC gene codes for the PTC taste receptor protein on the tongue How do genes affect phenotype? What is the structure of DNA?


Who discovered the structure of DNA? DNA Vocabulary to know:
DNA
Chromosomes
Genes
Alleles
The Central Dogma
Punnett Square
Dominant
Recessive
Genotype
Phenotype Watson and Crick Wilkins and Rosalind Franklin Linus Pauling DNA tells your body which proteins to make
-Proteins are molecules that can perform a variety of functions
-Structural Proteins
-Enzymatic Proteins
-Receptor Proteins Gregor Mendel’s peas plants (mid 1800’s)
Assumptions made:
For each trait (gene), there are two factors (alleles)
Some alleles are dominant and others are recessive
Each Parent gives its offspring exactly one allele t T T t Tt Tt Tt Tt DNA: Deoxyribonucleic acid; the “blueprint” for a person’s proteins. Gene: A sequence of DNA that codes for a specific protein Allele: Alternate forms of a gene The Central Dogma: DNA to RNA to Protein Punnett Square: will show the expected results of a genetic cross Dominant: An allele that will mask other alleles for that trait (A)
- Will be expressed if inherited (Aa or AA) Recessive: An allele that is masked by a dominant allele for that trait (a)
- Must be inherited by both parents in order to be expressed (aa) Homozygous: AA or aa Heterozygous: Aa Genotype: The genetic makeup of an organism Chromosome: An organized structure of DNA and proteins 8. Experiment (Steps 8-9) 9. Start PCR Lab 1. Brief Review and Introduction 3. PCR Discussion 5. Restriction Digest Discussion 7. Gel Electrophoresis Discussion
-Practice loading gels 4. Experiment (Steps 1-2) 6. Activity -Gel is a matrix, like tangled string

-DNA molecules can move through gel

-Small pieces of DNA can move quickly

-Larger pieces of DNA get more tangled in the gel and move more slowly

-Gel is made of agarose, from algae

-The smaller, faster molecules will move farther down the gel than the larger, slower ones

-By comparing location on gel to ladder of DNA of known lengths we can determine the length of our DNA strands Gel Electrophoresis Can be used to separate strands of DNA by size
Gel and electric current (positive and negative ends of gel)
Is DNA positively or negatively charged?
Which direction should the DNA move on the gel? Gel Electrophoresis We use micropipettes to load our DNA samples into the wells of the gel
We use a dye so we can see the liquid Gel Electrophoresis -Nontasters: no target region, one long uncut DNA fragment (303 base pairs)

-Homozygous tasters: only cut DNA, two different length fragments (238 and 65 bp)

-Heterozygous tasters: some cut DNA & some uncut DNA, all three lengths (303, 238, and 65 bp) Results of Electrophoresis Results of Electrophoresis 1, 4, 6, 9- heterozygous tasters Results of Electrophoresis Restriction Enzyme Digest Restriction enzymes cut DNA molecules A restriction enzyme recognizes
a unique sequence of nucleotides in DNA Restriction enzymes were originally found in bacteria as a part of their immune system We are using the restrition enzyme:
Fnu4HI Fnu4HI recognizes the sequence: 5' - GC NGC - 3' 3' - CGN CG - 5' The PTC taster gene has
been isolated and copied using PCR There are 3 nucleotide differences that make this gene code for a protein that cannot bind properly to PTC to give the bitter taste sensation Tasters will contain an Fnu4HI site Non-tasters will not contain the Fnu4HI site What does this Tasters will have
their DNA cut by the restriction enzyme Non-tasters DNA will remain the same length Non-taster DNA = 303 base pairs

Homozygous Taster = 238 + 65 base pairs

Heterozygous Taster = 303 + 238 + 65 base pairs What is a Pipet? Different Parts of a Pipet Using a Pipet Practice Pipetting twist knob to set amount
put on a clean tip
press plunger down to first stop
insert tip into liquid
release plunger slowly
remove tip from liquid A pipet is a device used to accurately measure and transfer small amounts of liquid in a sterile way To take up liquid: press plunger down past first stop all the way to second stop
remove tip from liquid before releasing finger To dispense liquid: -p10, p20, p200, p1000 Variety of sizes Tips -Pipet tips are where the liquid is taken up. They are sterile and are made to fit the different pipet sizes p-20 2 Volume Adjustment -Knob on the side allows you to set specific measurement 0 0 Volume Readout -Shows the set volume on the pipet (uL) tens ones tenths Plunger -Large button on the top allows you to draw up and dispense liquid Tip Eject Button -Pressing this button ejects the tip Practice Problems! - 1uL
- 6.5 uL
- 10 uL 2. Review of DNA Structure What is happening in the tube? Water: causes cheek cells to burst
Proteinase K: breaks down proteins of cheek cells to release DNA
Chelex buffer: attaches self to broken down proteins to separate from DNA Practice Loading Gels -Set pipet to 15 uL
-Get a clean tip
-Press plunger down to first stop, take up liquid
-At gel use two hands and steady your elbows
-Carefully insert tip into well, just past the surface of the liquid (be careful not to puncture the gel)
-SLOWLY push down plunger only to FIRST STOP to dispel liquid
-Remove tip from well before releasing plunger DNA is negatively charged so it will move from the negative end to the positive end of the gel. 2, 3, 8- nontasters 5- homozygous taster 7- blank 10- ladder Restriction Enzyme Activity Using scissors, pretend you are doing the job of the restriction enzyme and cut the DNA sequences at any Fnu4HI sites in the sequences mean? PCR Overview PCR: Annealing

1. Separate DNA strands using heat PCR: Denaturing
-Quick/selective way to prep DNA when the DNA source = insufficient or impure

-Any DNA segment can be targeted and amplified

-Billions of copies made of target segment in a few hours

Analogy: photocopying one page from a book rather than checking out the book from a library PCR Polymerase Chain Reaction (PCR) : Law of Dominance Law of Segregation Law of Independent Assortment Restriction Enzyme Activity Line up linking arms-you are base pairs in DNA!

Someone is a restriction enzyme...
Find the target sequence and cut the DNA (seperate arms). 8. Activity Up next...
how can we visualize this? = Nucleotide
Difference Is this allele
a taster (T)
or non taster (t)? = Fnu4HI Site Where is the DNA?


Why can we see it? Materials 1. Micropipette 2. PTC Paper 3. Centrifuge 4. PCR Machine 2. Cool down to allow hydrogen-bonding of single-stranded DNA primers What is in the tubes? Cheek Cell Tube: PCR Tube: DNA (from cheek cells)
Chelex buffer
Proteins from cell What are Other Mendelian Traits? PCR: Extension Water
New nucleotides
MgCl2
10x PCR buffer
Forward primer
Reverse primer
Taq DNA Polymerase 3. DNA polymerase adds nucleotides to 3'end of each primer Safety a. b. c. Wear goggles and gloves if you would like to

Don't eat or drink

Don't put anything in your mouth other than the PTC paper and the disposable loops

Be careful with the equipment

Don't chew gum-mint interferes with the experiment d. Day 1 Day 2 At Smith 1. PTC Taster Phenotype
2. DNA Extraction
3. PCR 4. Restriction Enzyme Digest 5. Gel Electrophoresis Day 1 Day 2 At Smith 1. PTC Taster Phenotype
2. DNA Extraction
3. PCR 4. Restriction Enzyme Digest 5. Gel Electrophoresis Day 1 Day 2 1. PTC Taster Phenotype
2. DNA Extraction
3. PCR 4. Restriction Enzyme Digest 5. Gel Electrophoresis You Are Here You Are Here You Are Here Day 1 Day 2 At Smith 1. PTC Taster Phenotype
2. DNA Extraction
3. PCR 4. Restriction Enzyme Digest 5. Gel Electrophoresis You Are Here What did we just do? Vortex Centrifuge Boil mix pellet denature proteinase K Why? What do you notice about each cross? *Extra* PCR vs. Natural Replication Natural Replication double-stranded DNA unwound
each strand = template
dNTPs: DNA building blocks
polymerase
end product: double-stranded DNA PCR naturally occuring
new DNA molecue formed
DNA helicase to separate strands
enzymes to carry out replication process unnatural/ performed in lab
targets specific region defined by primers
separate strands using heat
varying temperatures (72°C, 94°C, 55°C) Restriction Enzyme Activity
Part 2 Use your paper cutouts to simulate running a gel. 1 2 3 4 5 6 7 8 9 10 100 bp 200 bp 300 bp 400 bp cell to nerve to brain
to taste perception Activity Recap What does each person represent?


What do the groups of people represent?


What does the classroom represent? Nucleotide DNA Fragments The Gel PCR vs. Natural Replication Natural Replication double-stranded DNA unwound
each strand = template
dNTPs: DNA building blocks
polymerase
end product: double-stranded DNA PCR naturally occuring
new DNA molecue formed
DNA helicase to separate strands
enzymes: to carry out replication process unnatural/ performed in lab
targets specific region defined by primers
separate strands using heat
varying temperatures (72°C, 94°C, 55°C) What will this look like on a gel? Example Gel: Wells Ladder TT Tt tt 300 bp 200 bp 50 bp 5' - GC NGC - 3'

3' - CGN CG - 5' At Smith: Restriction Enzyme Activity Subject 1: tt, non-taster
Each chromosme = 1 fragment
Each chromosme = 31 nucleotides 9. At Smith Subject 2: Tt, taster
Chromsome #1 = 2 fragments
2 fragments
24, 7 nucleotides
Chromosome #2 = 1 fragment
1 fragment
31 nucleotides Subject 3: TT, taster
Each chromosome = 2 fragments
Each chromoseom = 24, 7 nucleotides 10. Analyze your gel results! Fnu4H1
restriction enzyme At Smith 6. Determine Your Genotype On Your Own 6. Determine Your Genotype On Your Own 1, 4, 6, 9- heterozygous tasters Results of Electrophoresis 2, 3, 8- nontasters 5- homozygous taster 7- blank 10- ladder 1 2 3 4 5 6 7 8 9 10 6. Determine Your Genotype On Your Own 6. Determine Your Genotype On Your Own 5' - GCNGC - 3'

3' - CGNCG - 5' + - 1 2 3 Subject # Double Helix Ladder Non-
Taster Heterozygous
Taster Homozygous
Taster -glowing bars -gel stained with dye that
binds to crevices of
double helix
-glows under UV light
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