Using a Single-Nucleotide
Polymorphism to Predict BitterTasting Ability
Carolina Kit
Timeline
Thursday—Lecture, volunteer aliquot
Monday—procedures quiz, Bioinformatics
HW: Bioinformatics (use website, not packet)
Tuesday—isolate DNA cells, amplify DNA
(PCR)
Wednesday—Volunteer pour gels
Thursday—digest samples, run gel,
photograph gel
Tuesday—Lab write-up due (after break)
Write-up
• Annotate handout
• Data
draw a gel and mark each banding site,
staple picture to lab that you turn in to me
• Results and Discussion—answer all parts
• Bioinformatics worksheet
Background Information
• http://bioinformatics.dnalc.org/ptc/animatio
n/ptc.html
read introduction
Single nucleotide polymorphism
DNA Science textbook: page 296-297
• Point mutation
• Most mutations are rare in a population, so to be helpful,
SNPs must have a population frequency of 1%
• A region of linkage is called haploblock because it is
inherited without recombination like haploid in mDNA
• A set of SNPs, markers, within the haploblock are
inherited as a haplotype.
• Different populations inherit different SNPs with the
haploblock
• This info. is great for linkage studies
• The hope is to make a map, find disease genes in
populations of unrelated people
Genotype and Phenotypes
• The TAS2R38 polymorphism was
specifically selected to demonstrate the
relationship between genotype and PTCtasting 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.
Prep. For lab--SNP
Week before
•
Label tubes
•
Pre-set thermo-cycler
By Tuesday
•
10 mL of .9% NaCl solution (.9g NaCl/100ml water) in 15 mL plastic tube (15)
•
100 uL 10% chelex into 1.5mL tube (15)
•
22.5uL of PTC primer/loading dye (30)
•
10uL of Restriction enzyme HaeIII (15)
•
20uL pBR322/BstNI marker (8)
•
paper cups
•
TBE 20x dilute to 1x to use (150mL TBE with 2850mL dwater)
Tuesday
•
Ice buckets with ice
Wednesday
•
Pour 2% gels, add ethidium bromide (200ng/mL final or 1uL of 10mg/mL stock in gel prepared
from 50mL), 6 well comb, TBE buffer
(10 grams agarose add up to 500mL TBE buffer)
•
Prepare UV trans. and camera
By Thursday
•
Set-up water bath 37 degrees
Preparing gels
•
•
•
•
•
•
___ grams agarose
Add up to ___mL buffer
Melt in microwave, let cool
Set up trays—use 6 well comb
Add 1uL ethidium bromide/50uL of solution
Pour about 30-50mL into each tray
Protocol
• http://bioinformatics.dnalc.org/ptc/animatio
n/ptc.html
review flow chart
Lab Day 1
Part I: isolate DNA
Part II:PCR
• We are doing cheek cells
• Work with a partner in your group
(15 sets in the class)
• we will use the heat block at set 9
• No Mineral oil for PCR
• I will store your PCR samples in the
freezer after PCR
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 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
results
• http://bioinformatics.dnalc.org/ptc/animatio
n/ptc.html
review results section
Bioinformatics
• http://bioinformatics.dnalc.org/ptc/animatio
n/ptc.html
Use website directions as it is most updated
• Complete the worksheet for homework