Download 1.PtII.SNPs and TAS2R38.v3

Using Single Nucleotide
Polymorphism (SNP)
to Predict
Bitter Tasting Ability
Part II: !
Digestion and Analysis of an Amplified Region
of the Bitter Taste Receptor TAS2R38 Gene
In The Last Lab:
•! You sampled some of your
own DNA and amplified a
short region of chromosome 7.
•! This region codes for part of
TAS2R38 gene, bitter taste
receptor gene.
In Today’s Lab:
•! You will digest some of your amplified PCR
product with the restriction enzyme, Hae III
•! And use agarose gel electrophoresis to
determine your genotype
•! You will also taste a sample of PTC paper and
determine your phenotype
•! You will compare the results of your genotype
with those of your phenotype – record on
board
•! Finally, you will use class data to determine if
the class is in Hardy-Weinberg equilibrium
Digestion of TAS2R38 With
Restriction Enzyme, Hae III
Agarose Gel Electrophoresis
Digesting PCR Sample with HAE III
Get your PCR tube from your instructor or TA (need your code)
•! Transfer 16 µl (0.016 ml) of your PCR product (amplified DNA) to a
flat top PCR tube
•! Label this tube “D” for “digested” and add your code.
•! This DNA sample will be digested with the restriction enzyme,
HaeIII
•! Ask your instructor or TA to add 1µl (0.001 ml) of the restriction
enzyme, HaeIII, to the “D” tube
•! Place this tube in the thermal cycler set for 37oC
and incubate for at least 30 minutes.
Label your original PCR tube with a “U” for
undigested; leave this sample on ice while the
other sample is incubated with HaeIII. !
http://highered.mcgraw-hill.com/olc/dl/120078/bio37.swf
http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#
(choose restriction endonucleases)
Agarose Gel Electrophoresis of
Amplified PCR Products
•!You will share an agarose gel (2%) with 2 other people
•!Load both the undigested and digested samples in
separate wells of the gel
•!Undigested - 10 µl
•!Digested - 16 µl
•! Also load the Marker, a 100bp ladder, in both end lanes of
the gel
•!
http://www.sumanasinc.com/webcontent/animations/content/gelelectrophoresis.html
•! http://www.wellesley.edu/Biology/Concepts/Html/gel.html
Agarose Gel
Run the gel for 30-40 minutes at 130 Volts; remove gel
and stain it with ethidium bromide
Staining DNA - Ethidium Bromide
•!Bring your gel to your TA who
will transfer it to the plastic
container and add ethidium
bromide stain to the DNA bands
•!Ethidium bromide intercalates
between the base pairs of the
DNA ladder
•!Stain the gel ~20 minutes
•!Destain ~ 10-15 minutes
•!View gel using UV light to make
the bands fluoresce
•!Analyze photo; attach it to your
worksheet
Determining Your Genotype
http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#
Choose RFLP-restriction fragment length polymorphism
Determining your PTC Phenotype
•!
•!
•!
•!
Taste the Control paper first (no PTC)
Taste the PTC paper
Place strip in your mouth for a few seconds
Remove it and wait; it takes a few seconds
to taste PTC
•! Record both your genotype and phenotype
on board
•! You will need these results to complete the
post-lab assignment
Class Results
Genotype
Phenotype
Phenotype
strong taster
weak taster
Phenotype
non-taster
TT
Tt
tt
* Record the class results on your worksheet.
Worksheet
•! Do sample calculations
•! Calculate expected allele and genotype
frequencies using the sample data
given:
•! T/T: 15
•! T/t: 50
•! t/t: 35
•! Do Chi square calculation to determine
if the sample population is in HardyWeinberg equilibrium
http://www.pbs.org/wgbh/nova/sciencenow/0302/01.html (12minutes)
Personal DNA Testing
Next Lab
DUE: C-fern Lab Report
Pre-lab on RPI-LMS:
Genetics & Medicine Simulation Lab:
Sickle Cell Alleles
Quiz: Using SNPs to Predict Bitter Tasting Ability
•! How well does TAS2R38 genotype predict PTC-tasting
phenotype?
•! What does this tell you about classical dominant/recessive
inheritance?
The presence of a T allele generally predicts tasting, although
heterozygotes are more likely to be weak tasters. Even in a relatively
simple genetic system such as PTC tasting, one allele rarely has complete
dominance over another. This experiment examined only one of several
mutations in the TAS2R38 gene that influence bitter tasting ability.
Variability in taste perception is likely affected by processing in the brain,
which involves numerous other genes. (E.G., There are about 25-30 genes
that code for bitter taste receptors. Also the genes for three of the cranial
nerves involved in taste perception.) Thus, perception of bitter tasting
substances like PTC is complex and does not exactly follow the simple
Mendelian inheritance pattern.
The forward primer incorporates part of the HaeIII
recognition site, GGCC. How is this different from the
sequence of the human TAS2R38 gene?
Mismatch between
forward primer & alleles
Formation of HaeIII Recongition Site
in Amplified PCR Product
http://www.pbs.org/wgbh/nova/body/tongue-taste.html
Making sense of SNPs and
the Albuterol Story
http://www.genome.gov/Pages/Education/DNADay/
TeachingTools/MakingSNPsMakeSense.html