1. Lab 8: PTC Polymerase Chain Reaction Lab
Using a Single-Nucleotide Polymorphism (SNP) to Predict Bitter-Tasting Ability

2. PTC Polymerase Chain Reaction Lab
Notebook
PTC Polymerase Chain Reaction Lab
Objectives
To understand taste perception of the PTC gene and how SNPs can be used as molecular markers to predict phenotypes
To become acquainted with modern molecular genetics techniques used in SNP mapping that include: DNA extraction and purification, polymerase chain reaction (PCR), DNA restriction and gel electrophoresis

3. PTC Gene (Bitter Tasting Ability)

4. PTC Gene
Ability to taste PTC (phenylthiocarbamide) is an inherited dominant trait
Varies in the human population and influences taste
PTC taste receptor gene is TAS2R38 (located on chromosome 7) – bitter tasting ability

5. PTC gene is just one of 30 bitter taste receptors on the tongue

6. PTC Procedure Collection and Isolation of DNA From Your Cheek Cells
Notebook
PTC Procedure
Collection and Isolation of DNA From Your Cheek Cells
Amplifying PTC DNA by Polymerase Chain Reaction (PCR)
Restriction Digest of PCR Products with HaeIII (Restriction Enzyme)
Gel Electrophoresis of a Restriction Fragment Length Polymorphism (RFLP)

7. Single Nucleotide Polymorphism (SNP)
SNPs are common genetic variations
A SNP represents a variation in a single nucleotide of a gene
Use HaeIII restriction enzyme to recognize the SNP sequence of the PTC gene
GGCC

8. Single Nucleotide Polymorphism (SNP)
Identify subtle differences in DNA and how they affect you

9. Are You a Non-Taster, Weak Taster, or a Strong Taster?
Notebook
Are You a Non-Taster, Weak Taster, or a Strong Taster?
Control Paper
PTC
Non-Taster
PTC Weak Taster
PTC Strong Taster
You
Class

11. Part A: Collection and Isolation of DNA From Your Cheek Cells
Swab inside of mouth to obtain cheek cells
Rinse cells in saline
Centrifuge to obtain the cells in a pellet
Use Chelex resin (beads) to extract DNA
Heat on a block to melt phospholipids and denature proteins

12. Part B: Amplifying PTC DNA by Polymerase Chain Reaction (PCR)
Notebook
Part B: Amplifying PTC DNA by Polymerase Chain Reaction (PCR)
1. Add PCR Master Mix (22.5 uL) to your DNA (2.5 uL) in PCR tube
Gene specific primers – locate the PTC gene
dNTPs (mixture of A T C G nucleotides)
Salts – neutralize the electrically charged sugar phosphate backbone
pH of 8.9
One Taq DNA Polymerase
2. Perform PCR Amplification using miniPCR Thermocycler

13. Amplifying PTC DNA by Polymerase Chain Reaction (PCR)

15. Part C: Restriction Digest of PCR Products with HaeIII
Notebook
Part C: Restriction Digest of PCR Products with HaeIII
1. Label two 1.5 ml microcentrifuge tubes – one “U” for undigested and one “D” for digested - Label both tubes CLEARLY with your initials 2. Add the following to the tubes:

16. Part C: Restriction Digest of PCR Products with HaeIII (continued)
Notebook
Part C: Restriction Digest of PCR Products with HaeIII (continued)
3. Centrifuge the “D” tube to pool reagents 4. Place “D” tube in a 37°C water bath for 15 minutes 5. Store in freezer until gel electrophoresis experiment

17. Electrophoresis Prediction
Notebook
Electrophoresis Prediction
Based on the results of the PTC taste test (phenotype), draw your predicted electrophoresis results
Label each band on the gel with the appropriate fragment size in base pairs

18. Notebook
Part D: Gel Electrophoresis of a Restriction Fragment Length Polymorphism (RFLP)
1. Add 2 µL of loading dye to the “U” tube and the “D” tube. 2. Using a p20 micropipette, load 18 µL of each sample into each well. 18 µL of DNA ladder will also be loaded into a well. 3. Record the well number(s) you loaded your samples into the gel. 4. Run the gel at 100 volts for 30 minutes and observe gel under UV light. Take a picture of the final gel.

19. What will be visible on the gel?

20. Gel Electrophoresis Results
Notebook
Gel Electrophoresis Results
Draw a picture of the gel electrophoresis results for your samples (U and D) into your lab notebook. Indicate the size of each fragment in base pairs (bp)