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Carolina: Using SNP’s to Predict Bitter-Tasting Ability (PTC) - CHECKLIST
Name:_____________________________________________Date:______________
INTRODUCTION:
In the early 1930’s, Arthur Fox had synthesized some phenylthiocarbamide (PTC) and some of the
PTC dust escaped into the air as he was transferring it into a bottle. Lab-mate C.R. Noller complained the
dust had a bitter taste, but Fox tasted nothing. The inability to taste PTC is a recessive trait that varies in the
human population.
Bitter-tasting compounds are recognized by receptor proteins on the surface of taste cells. There are
approximately 30 genes for different bitter taste receptors in mammals. The gene for the PTC taste receptor,
TAS2R38, was identified in 2003.
Polymerase Chain Reaction (PCR) is used to amplify a short region of the TAS2R38 gene. The
amplified PCR product is digested with the restriction enzyme HaeIII (Haemophilus aegyptius – causative
agent of conjunctivitis, also called pink eye) whose recognition sequence includes a Single-Nucleotide
Polymorphism (SNP); in this case, HaeIII will identify the G-C polymorphism in the TAS2R38 gene
(Recognition site: GGCC). Nontasters will produce an uncut 221 bp fragment (GGGC). Tasters (GGCC)
will produce 2 fragments: 44 bp and 177 bp. One allele is cut by the enzyme, and one is not – producing a
Restriction Fragment Length Polymorphism (RFLP).
In the concept of pharmacogenetics, SNP genotypes are used to predict drug responses.
LAB:
Student Supplies:
2 - 1.5 ml tube
*Prepared Reagents:
1- paper cup
2 – micropipettors (p20 & p200)
1 – 15 mL conical tube to transfer saline
Permanent marker
- 10 ml 0.9% saline /person; prepare in 50 mL conical tubes
- 100 µL of 10% Chelex, in 0.5 ml tube or screwcap tube
(whichever is available)
Part I. Cheek Cell DNA Isolation
_______1) Prepare 0.9% saline solution using distilled water using 50 mL conical tubes (10 mL/person
required).
_______2) Transfer 10 mL of saline into 15 mL conical tubes for dispensing into cups.
_______3) Label 1.5-ml tube and paper cup w/ your assigned #.
_______ 4) Dispense saline from conical tube to your paper cup and return empty conical tube to teacher
station.
_______ 5) Pour saline into your mouth and vigorously rinse for 30 seconds.
_______6) Expel saline into your paper cup.
_______7) Swirl cup gently, then using a p200, transfer 1000μL of solution into your 1.5 mL tube. Don’t
dispose of paper cup until after step #9.
_______8) Spin in balanced microfuge for 90 seconds at full speed.
Princeton Satellite Site
Spring 2011
K. McKone
Carolina: Using SNP’s to Predict Bitter-Tasting Ability (PTC) - CHECKLIST
Supernatant = liquid above pellet
*DNA is in the pellet!
_______9) Carefully pour off supernatant into your paper cup. Try to remove most of supernatant but be
careful not to disturb pellet. (Remaining volume will reach ~ 0.1 mark of tube). *Now you may throw away
paper cup if work area is crowded.
*Optional: Collect small sample of cells to observe under microscope.
_______ 10) Set micropipette to 30μL. Resuspend cells in remaining saline by pipetting in and out. Be
careful to minimize bubbles.
_______ 11) Withdraw 30 μL of cell suspension, and add it to a screwcap tube containing 100 μL of Chelex.
Label cap and side of tube w/ assigned number. May dispose of used 1.5 mL tube.
_______12) Boil for 10 minutes @ 99oC.
________13) Shake tube vigorously for 5 seconds.
___ ____14) Spin for 90 seconds at full speed.
_______ 15) Transfer 30 μL of clear supernatant into CLEAN 1.5 mL tube. Avoid Chelex beads or cell
debris.
_______16) Label w/ assigned number.
_______ 17) Store @ -20o C until you are ready for PCR.
Part II. Amplification: Amplify DNA by PCR. Thermal Cycler PREP
Reagents:
PTC primer/loading dye mix - 22 µL
Ready-To-Go Beads
Cheek cell DNA
Equipment:
Microcentrifuge tube rack
Thermal cycler
Crushed ice recommended but may be omitted
_____1) Label a PCR Ready-To-Go tube.
_____2) Make sure PCR bead is at the bottom of PCR tube, then add:
Primer solution – 22
(Forward Primer) 5’- CCT TCGT TTT CTT GGT GAA TTT TTG GGA TGT AGT GAA GAG GCG G-3’
(Reverse Primer) 5’ – AGG TTG GCT TGG TTG CAA TCA TC-3’
Your DNA – 3 µL
_____ 3) Mix by tapping sample on table (or VORTEX briefly) & PULSE SPIN
_____4) Place tube in thermal cycler and document location.
Princeton Satellite Site
Spring 2011
K. McKone
Carolina: Using SNP’s to Predict Bitter-Tasting Ability (PTC) - CHECKLIST
Back of THERMAL CYLCER; Princeton holds 20 samples ~ 1Hour 30 Minutes
Left
Right
Front
**PCR Protocol for Programming Thermal Cycler: Takes ~ 1 hour 30 minutes.
Cycle
Step
30x
1
2
3
Ftn.
Temp.
Denature
Anneal
Extend
94oC
64oC
72oC
Time
30 sec
45 sec
45 sec
*If you won’t be around when it finishes, put on hold @ 4oC. Store on ice until the day arrives to use.
Part III. Digest PCR Products with HaeIII.
Reagents:
Supplies:
-PCR products from Part II (25 µL)
-Restriction Enzyme HaeIII;
1 µL/ student
--1.5 mL microfuge tube
-p20
-p10 (if available)
-Water bath or heat block @ 37oC for @ least 30 minutes;
1 hour is better.
_______1) Label the clean 1.5 mL tube with your assigned number and with a “U” (undigested).
_______2) Use a micropipette with a fresh tip to transfer 10 µL of your PCR product to the “U” tube.
Store in the freezer until you begin Part IV.
_______3) Add 1 µL of restriction enzyme (HaeIII) directly into remaining PCR product in PCR tube. Label
tube with “D” (digested).
_______4) Pulse spin or tap tube on lab bench.
_______5) Incubate the “D” tube at 37oC for at least 30 minutes (time permitting; incubate for 1 or more
hours).
Part IV. Prepare for E-Gel Electrophoresis (This requires UV illuminator):
Reagents:
Undigested PCR product “U”; 10 µL
HaeIII-digested PCR product “D”; 16 µL
pBR322/BstNI marker
Equipment:
2% E-gels
UV illuminator
*If UV illuminator is unavailable, make 2% agarose gels.
______1) After removing your PCR products from thermal cycler, tap tube on table.
Princeton Satellite Site
Spring 2011
K. McKone
Carolina: Using SNP’s to Predict Bitter-Tasting Ability (PTC) - CHECKLIST
No Loading Dye necessary b/c Primer mix contained cresol red dye.
______2) Obtain e-gel for loading.
______3) Plug E-Gel PowerBase into electrical outlet.
______4) Insert gel (with comb in place) into the base RIGHT edge first.
______5) Conduct 2 minute Pre-Run by holding down either button until red light starts flashing green.
______6) Remove comb from E-Gel cassette and place in cassette wrapper for disposal.
______7) Each well may hold up to 20 µL but our samples aren’t that large. Load the full amount of your
samples: 10 µL of “U” and 16 µL of “D”.
______8) Load water into any remaining empty wells.
______9) Run for 30 minutes.
E-gel Loading Order – Complete the following chart. Identify student “U” & “D”.
Don’t forget 2 minute Pre-Run!
1
ID
Volume
µL
Results
# of
Bands
2
3
4
5
Marker
6
7
Marker
8
9
10
11
12
15 µL
Total Gel Run Time: ______________ minutes
Genotypic Analysis:
- tt nontaster (homozygous recessive) = a single band (221 bp) in the same position as the uncut control.
NONTASTER
- TT taster (homozygous dominant) = 2 bands ( 177 & 44 bp). Incomplete digestion may leave a small amount of
uncut product at the 221-bp position, but is should be fainter than the 177-bp band. STRONG TASTER
- Tt taster ( heterozygous) = 3 bands representing both alleles – 221 bp, 177 bp, and 44 bp. The 222-bp band must be
stronger than the 177-bp band. (If the 221-bp band is fainter, it is an incomplete digest of TT.
Phenotype
Strong taste
Weak taste
Nontaster
TT
Tt
tt
Students
Genotype
Students
Princeton Satellite Site
Spring 2011
K. McKone