1.16: A Taste of Genetics - PTC Taster

Learning Objectives

Goals:

• To understand basic PCR and gel electrophoresis
• To understand basic DNA mutation detection
• To learn how restriction enzymes are incorporated in biotechnology

Student Learning Outcomes:

Upon completion of this lab, students will be able to:

• Understand SNPs
• Know how to perform a DNA extraction, PCR, and restriction digest
• Know how to interpret a DNA gel after electrophoresis

Introduction

Every organism on Earth has a different way to perceive the world due to their individual life experiences as well as their genetic make-up. Humans are no different; every individual has their own experiences that shapes their world perception but so too does their DNA. You may be surprised to learn that, 99.9% of the human genome is identical from one individual to the next, and it is the 0.1% difference that makes each individual unique.

Some of these differences can affect our sensory systems and how we perceive the natural world. For example, over time we have learned which things taste good and are good for us while simultaneously learning which things taste bad or are bad for us. Specifically, bitter compounds are closely associated to toxic substances in nature. The way we know things taste bitter, or any other flavor for that matter, is because we have special chemical receptors in our mouth and nose that bind molecules in our food and send signals to the brain telling it what the food tastes like.

Figure 1: A chemical binding a membrane receptor

One type of bitter receptor in our mouth senses the presence of a chemical called phenylthiocabamide, or PTC. PTC is a non-toxic chemical but it very closely resembles toxic compounds often found in food. The unique thing about PTC is that not everyone can taste it! We first learned this in the 1920s when Arthur L. Fox and C. R. Noller were working with PTC powder and Noller complained about the extremely bitter taste while Fox tasted nothing at all. This lead to experimentation where scientists ultimately discovered the ability to taste PTC was hereditary; it was in our DNA!

Before we talk about the genetics of PTC tasting, we first need to understand some terminology. The observable trait, such as the ability to taste PTC, is called a phenotype. The genetic information that codes for that phenotype is called a genotype. The genes that make up a genotype come from the parents in the form of alleles; one allele from the mother and one allele from the father. The two copies can be the same allele, homozygous, or the two copies can be different, heterozygous.

The ability to taste PTC comes from the gene TAS2R38 which encodes one of the chemical receptors in our mouth that binds to PTC. By comparing PTC tasters to non-tasters, scientists have found three single nucleotide polymorphisms (SNPs) that differentiate the taster allele (T) from the non-taste allele (t). A SNP is a genetic mutation where one nucleotide in DNA is different from one individual to the next. The word mutation sounds scary but a mutation is not always bad; there are nearly 10 million SNPs in humans which means SNPs are common. The three SNPs (see table 1) found in the TAS2R38 gene leads to changes in the amino acid sequence which can potentially change the proteins function.

Table 1. SNPs Present in Tasters vs Non-Tasters for PTC

Nucleotide position (bp)	Nucleotide Change		Codon Change		Amino Acid Change
	phenotype	Non-Taster	Taster	Non-taster	Taster	Non-taster	Taster
145	G	C	GCA	CCA	Alanine	Proline
785	T	C	GTT	GCT	Valine	Alanine
886	A	G	ATC	GTC	Isoleucine	Valine

Before you figure out your tasting ability, lets first understand the genetics of the alleles. Individuals who are tasters can be TT (homozygous dominant) or Tt (heterozygous). Individuals who are non-tasters will always be tt (homozygous recessive). To understand how the genes are inherited, examine table 2 below where the potential offspring of two heterozygous parents are analyzed. There is a 75% chance of having children that are tasters for PTC and a 25% chance of having children that are non-tasters.

Table 2. Sample Inheritance Pattern for PTC Tasting

Parent Alleles	T	t
T	TT (homozygous taster)	Tt (heterozygous taster)
t	Tt (heterozygous taster)	tt (homozygous non-taster)

We will figure out your genotype today using three very commonly used assays in the field of biotechnology. The first is polymerase chain reaction (PCR) which is used to selectively amplify a specific region of DNA of interest. PCR allows us to take one or two copies of DNA and make millions of them making it easier for us to analyze the results. Then we will perform a restriction digest with restriction enzymes. Restriction enzymes are like “molecular scissors” because they cut DNA at specific nucleotide sequences called recognition sites. For this lab, you will be using the restriction enzyme called HaeIII, which recognizes the sequence GGCC. When HaeIII comes across the recognition sequence, the enzyme will cut the DNA between G and C nucleotides producing two different size DNA strands. In order to visualize the DNA, we will run gel electrophoresis, our third assay, which allows us to separate DNA molecules based on their size. See Figure 2 below for the expected results.

Figure 2:. Expected gel electrophoresis results post-restriction digest

Part I: Day 1

Materials

Figure 3. Day 1 Reagents

Reagents

• PTC and control paper strips
• 2 Small microcentrifuge/PCR tubes
• 0.9% saline solution
• Extraction solution
• Taq master mix
• Primer mix

Equipment

• P-20 and P-200 micropipettes and disposable tips
• Microcentrifuge
• Thermocycler 
• Ice bucket 
• Freezer -20^oC
• BioWaste container (for saliva, tips, test strips, PCR tubes)
• Rack for PCR tubes (microtiter plate or empty p_200 tip boxworks as susbstitute)

Procedure (per manufacturer guidelines)

1. Place one strip of PTC paper on the tip of your tongue and record whether it taste bitter or not. Discard the used PTC paper in biological waste
   • Bitter
   • Not Bitter
2. Tally the students in the class to determine the number of tasters and non-tasters and place that information in the box below:

Table 3. Class Data

Phenotypes	Number of Students	% Total
PTC Taster
PTC Non-taster
Total

3. Label 2 PCR tubes and a cup of saline solution with your own identifier/initials.
4. Pour the 0.9% saline solution into your mouth and swish vigorously for 2 minutes to dislodge the cells in your mouth. This is where the DNA will be coming from in our experiment.
5. Pipette 200µL of your saliva/saline mix into one of the labeled PCR tubes and close the PCR tube tightly.
6. Centrifuge the PCR tube containing the saliva/saline at 8,000RPM for 3 minutes. (Be sure to counterbalance the tubes).
7. Look for the white cell pellet at the bottom of the tube. Carefully remove the supernantant using a micropipette (do not disturb the pellet) and discard into a biological waste container. Be careful not to disturb the cell pellet!
8. Add 50µl of the extraction solution to the PCR tube with the cell pellet. Resuspend the cells by mixing using the micropipette and continue to do so until the cell pellet is broken up and there are no longer large clumps of cells.
9. You need to incubate your tube at 95°C for 5 minutes to break open the cells and release the DNA into solution, followed by cooling it until ready to use. You can place a tube on ice to chill it. If using the MiniOne system, place the tube in the PCR machine. Using the mobile device with MiniOne PCR mobile app, program the PCR machine using the constant temperature mode to incubate the samples at 95°C for 5 minutes. Enter 4°C for final incubation temperature. This will keep your samples cold until you are able to pick them up. (Table 4)

   Table 4 Cell Lysis Program

   Step	Duration	Temperature
   Cell Lysis	5 mins	95⁰C
   Final Incubation	∞	4⁰C

10. Retrieve PCR tubes and centrifuge for 1 minute at 8,000 RPM to collect cell debris at the bottom of the tube. Your DNA will now be found in the supernatant of the tube.
11. Without disturbing the pellet at the bottom, carefully pipette 5µL of the DNA containing supernatant into your 2^nd labeled PCR tube.
12. To your PCR tube containing DNA, add 10µL of Taq Master Mix and 5µL primer mix. Make sure to avoid placing a bubble at the bottom of the PCR tube as this can affect the PCR reaction.
13. Cap the tube tightly, gently flick tube to mix, then centrifuge for 15 second at 8,000RPM to bring all the liquid to the bottom of the tube.
14. Place the PCR tube in the thermocycler. When all samples are loaded, close the lid and follow instructor’s direction to set up the PCR protocol as seen in Table 16.5.
15. Once the protocol is complete, remove your sample from the thermocycler and place at -20⁰C until next class period.

 

Table 5. PCR Program (optimized for MiniOne PCR System)*

Step	Duration	Temperature	Cycles
Initial Denaturation	30 sec	94⁰C
Denaturation	5 sec	94⁰C	30 Cycles
Annealing	10 sec	66⁰C
Extension	15 sec	66⁰C
Final Incubation	∞	4⁰C

Part II: Day 2

Figure 4. Day 2 Reagents

 

MATERIALS

Reagents

• Stored frozen sample from previous period
• New PCR tube
• HaeIII restriction enzyme
• Dilution buffer
• Agarose gel with Gel Green
• Loading dye
• Running buffer (TBE)
• DNA marker

Equipment

• Water bath or programed thermocycler
• Gel casting tray and comb
• Gel electrophoresis unit
• Microcentrifuge
• Blue light box
• Photo documentation equipment

Procedure

1. Obtain your PCR tube from the previous lab session
2. Split your reaction into two by pipetting 10µL of your PCR product into a clean PCR tube. Label one “U” for undigested and the other “D” for digested.
3. Add 5µL of HaeIII restriction enzyme to the “D” tube and 5µL enzyme dilution buffer to the “U” tube. Cap the tubes and gently flick with your fingers to mix. Centrifuge your tubes for 15 seconds at 8,000 RPM to collect all liquid to the bottom of the tube.
4. Place your tubes in a suitable water bath or thermocycler using the settings in Table 5. When using the MiniOne PCR System, set up the incubation for the restriction digest at 37⁰C for 15 minutes using the constant temperature mode. Enter 4⁰C for the final incubation. (See table 6)

   Table 6. HaeIII Digest Program

   Step	Duration	Temperature
   HaeIII Incubation	15 mins	37⁰C
   Final Incubation	∞	4⁰C

5. While you wait for your digest, prepare an agarose gel. You will need a dye such as gel green included to visualize DNA in the gel. Lab 11 has more detailed instructions if you are not using the kit. For the MiniOne kit, the gel green is included in a premeasured amount of agarose. Poke a small hole in the plastic on top of the gel cup to allow for steam to escape. Microwave gels for 20 second increments until the gel is completely dissolved and in a liquid state. Pour your gel in the casting tray using the 9-well side of the comb. Allow your gel to solidify (it will be somewhat opaque when dry).
6. When incubation is complete, retrieve your samples. Add 3µL of loading dye to each of your tubes containing DNA. Cap your tube and flick gently to mix reagents. Centrifuge your tubes for 15 seconds at 8,000RPM to bring liquid down to the bottom of the tube.
7. Obtain your electrophoresis unit. For the MiniOne Gel Tank, ensure the black platform is in the tank to aid in visualization. Place your gel in the tank and ensure the wells are on the negative end of the gel box.
8. Pour TBE running buffer into the tank and ensure the gel is completely submerged by the buffer. Incomplete submersion of gel will lead to pour results in the gel electrophoresis.
9. Turn on the low intensity blue light and load 10 μL of your undigested sample and 10 μL of your undigested sample into two adjacent wells of the gel. Make sure your group also loads a DNA marker into one of the wells. Your group may use 10 μL of the MiniOne® DNA Marker. Use table 6 below to keep track of which samples are loaded into which wells.

   Table 7. Loaded Samples

   Well	1	2	3	4	5	6	7	8	9
   Sample

10. Turn on the MiniOne Electrophoresis System by placing the orange cover onto machine and pressing the power button. The green light should turn on and small bubbles should be visible in the buffer solution. Run samples for 20 minutes to allow proper separation of bands. If using a different electrophoresis system run the gel at 135V until the bands separate sufficiently and the dye front has traveled about 70% down the gel.
11. At the end of the run, turn on the high intensity blue light and use your phone, camera, or gel documentation system to take a picture of the gel. The blue light make the gel green that is incorporated into the DNA molecules fluoresce so they can be visualized.
12. Analyze the gel based on the information provided in the introduction of this lab.
13. Dispose of your gel and TBE buffer according to instructor instructions.

Study Questions

1. If someone can taste PTC, what is/are their possible genotype(s)?
2. If someone is homozygous for a trait versus heterozygous, when comparing their results on gel electrophoresis, what differences, if any, do you expect to see.
3. When you used PCR to amplify the TAS2R38 gene, what component of the reaction makes it specific for that gene in your genome and not another gene?
4. Restriction enzymes recognize very specific sequences in the DNA. They read the same forward and reverse. What are these types of sequences called?
5. If you did not see any bands in your reaction after electrophoresis, what might have gone wrong? List two possible reasons for this result.
6. After comparing your bands to those of the marker DNA bands, did your bands and those of your classmates match the expected size bands?
7. Do your results in the DNA band analysis match your phenotype as a taster or non-taster based on the paper taste? What did you expect to see for the different phenotypes in the class?

Attributions

This lab is licensed as CC BY-NC-SA. The title, figure 2 and procedure are taken from the lab developed by Embi Tec and used with permission.