Zusammenfassung der Ressource
2.1.3: Test Your Own Genes
- Isolate your DNA
- Amplify Your DNA by PCR
- 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