Using a Single Nucleotide
Polymorphism (SNP) to Predict
Bitter Tasting Ability
Carolina Kit
Timeline
• Day 1: Lecture
• Day 2: Isolate DNA, then amplify using
PCR methods
• Day 3: Pour gels (lab service)
• Day 4: Digest cells (lab service—1 from
each group—start at lunch), run and
photograph gel
Prep for Lab
Week before
•
Label tubes
•
Pre-set thermo-cycler
3 students at 8 stations will give their DNA
By Day 1
•
10 mL of .9% NaCl solution (.9g NaCl/100ml water) in cups (24)
•
100 uL 10% chelex into 1.5mL tube (24)
•
22.5uL of PTC primer/loading dye (24)
•
20uL pBR322/BstNI marker (8)
•
paper cups
•
TBE 20x dilute to 1x to use (150mL TBE with 2850mL dwater)
•
Ice buckets with ice
Day 2
•
Pour 2% gels, add ethidium bromide (200ng/mL final or 1uL of 10mg/mL stock in gel prepared
from 50mL), 10 well comb, TBE buffer
(10 grams agarose add up to 500mL TBE buffer)
•
Prepare UV trans. and camera
Day 3
•
Set-up incubator at 37 degrees
Background Information
• http://bioinformatics.dnalc.org/ptc/animatio
n/ptc.html
• Important Concepts
-SNPs
-DNA Isolation
-PCR
-Gel Electrophoresis
SNPs
• Single Nucleotide Polymorphisms
• http://www.youtube.com/watch?v=tJjXpiWKMyA
• For a variation to be considered a SNP, it must
occur in at least 1% of the population.
• SNPs, which make up about 90% of all human
genetic variation, occur every 100 to 300 bases
along the 3-billion-base human genome.
Relationship between Phenotype
and Genotype
• Relationship: The genotype ("internally coded,
inheritable information") codes for the phenotype
(outward, physical manifestation)
• The TAS2R38 polymorphism was specifically
selected to demonstrate the relationship
between genotype and PTC-tasting phenotype,
because it has no known relationship to disease
states or sex determination.
• TAS2R38 alleles are inherited in a Mendelian
fashion and can give indications about family
relationships
Lab Day 1
Part I: isolate DNA
Part II:PCR
• Cheek cells
• No Mineral oil for PCR
• Store your PCR samples in the freezer
after
Isolation
• From cheek cells
• Chelex will be the reagent used
• Boiling step: lyses the cell membrane ,
releasing DNA and other cell contents
PCR- Polymerase Chain Reaction
• Sections of DNA can be recognized and
reproduced
• Based on cell respiration (meiosis)
• Three steps:
1) Denature (seperation of two strands of
DNA) at 90 degrees
2) Anneal (binding of DNA primer to the
seperated strands) at 72 degrees
3) Elongate (elongation of the strands
using the DNA primer with heat stable
DNA polymerase) at 50 degrees
PCR tutorials:
http://learn.genetics.utah.edu/content/labs/pcr/
http://www.dnalc.org/resources/animations/pcr.html
Lab Day 2
Part III: Digest
Part IV: electrophoresis
• Make sure to label with a “D” and “U”
• At step 5, use the water bath instead of
thermo-cycler
• Skip step 9, we already added ethidium
bromide
• Test your bitter taste
Gel Electrophoresis
• A process that uses electricity to separate
charged molecules, such as DNA fragments,
RNA, and proteins, on a gel slab
• Larger sections of DNA is the slower it moves
through gel
• DNA is negative so it runs to the positive
• The buffer in the gel is conductive and allows
the electricity to flow through the gel and
stimulate the DNA
Gel Loading
1.
Marker
2.
Partner set 1-U
3.
Partner set 1-D
4.
Partner set 2-U
5.
Partner set 2-D
6.
Empty
Make sure to record
what is in each lane in
your lab notebook
•
Resultshttp://bioinformatics.dnalc.org/ptc/animation/ptc.html
Protocol
• http://bioinformatics.dnalc.org/ptc/animatio
n/ptc.html
Bioinformatics
• http://bioinformatics.dnalc.org/ptc/animatio
n/ptc.html
Use website directions as it is most updated
• Complete the worksheet for homework
Sources
• http://www.ornl.gov/sci/techresources/Hum
an_Genome/faq/snps.shtml
• http://www.brooklyn.cuny.edu/bc/ahp/BioIn
fo/GP/Relationship.html