Digest PCR Products with
Restriction Enzyme HaeIII
Analyze PCR Products by Gel
Electrophoresis
Determine Your PTC
Genotype and Phenotype
Observe the bands on gel & compare digested
DNA to uncut control and determine own
geneotype
Obtain PTC paper and test ability to taste the
chemical; correlate PTC genotype w/ phenotype
Gel Electrophoresis results: one band indicates recessive
alleles b/c no cut made by HaeIII (my results) meaning
unable to taste PTC
Homozygous Dominant: 2 bands resulting
from 1 cut by HaeII on both alleles
Heterozygous: 3 bands resulting from one
cut on GGCC allele and no cut on GGGC
allele
Goal: compare and correlate our genotype with
our phenotype
Place solidified gel in electrophoresis
chamber and add TBE buffer
Load the following into the gel:
Lane 1 - DNA Marker - 20ul
Lane 2 - Undigested DNA - 10ul
Lane 3 - Digested DNA - 15ul
Run gel at 130V for 30 min.
TBE Buffer: conducts electricity to
allow successful travel of bands
Goal: observe the separation of the bands in order to
determine genotype
Label 2 clean 1.5 ml tubes: "undigested" and
"digested"
Transfer 15 ul of PCR product into "digested"
tube and remainder of the product to
"undigested" tube
Pipet 1 ul of enzyme HaeIII directly into PCR
products in "digested" tube
Transfer digested sample to a .2ml tube
Incubate tube in thermal cycler for 1 hr
HaeIII: restriction enzyme that recognizes the
GGCC sequence of PTC and cuts between bp
to create a blunt end
Goal: to cut the DNA gene at the GGCC seequence and
understand difference b/w tasters & non-tasters
Obtain a PCR tube containing a PCR bead
Add 23 ul of PTC primer/loading dye mix to tube
Allow PCR bead to dissolve in the mix
Use micropipette to transfer 3 ul of the
cheek cell DNA into the mix
Place PCR tube in thermal cycler (programmed for
40 cycles)
After cycling, store the amplified DNA on ice
Steps of PCR: 1.
Denature at 94
degrees C for 30 sec
2. Anneal at 64
degrees C for 45 sec
3. Extend at 72
degrees C for 45 sec
Primer Loading Dye: adds the primers specific
to the TAS2R38 gene, needed to initiate elongation and
adds the color needed to see the bands during
gel electrophoresis
PCR Bead: contains the taq polymerase,
dNTPs , and buffer needed to amplify DNA
Goal: make numerous copies of onyl PTC gene
Spit into cup: .9% Saline w/
cheek cells
Take 100 ul and spin/centrifuge - allows
separation of cheek cells from saliva
Cells (w/ DNA in nucleus) clump at bottom
Pour off supernatant (spit)
Re-suspend cells: pipet up & down
Withdraw 30 ul of solution and add to tube
containing 100 ul of Chelex; heat tube for 10 min.
Centrifuge again - cell debris sinks to the
bottom; supernatant contains DNA
Transfer 30 ul of supernatant to new 1.5ml tube
Chelex: protects the DNA from the
heat of thermal cycler
Heat/Thermal Cycler: allows the cells to burst
and release the DNA
Goal: separate DNA from saliva and other
cell parts for amplification
Objective: Determine my genotype for the ability
to taste PTC and compare/connect it to my
phenotype