Download PCR

Dawid Potgieter 1st year Biochemistry
Aim: Use PCR to amplify the Ala nucleotide sequence on the TPA gene found on chromosome
8. Electrophoresis can then be used to determine the how many first year biochemists are
heterozygous and how many are homozygous.
Apparatus for gel electrophoresis.
Hypothesis: About 50% of people should have
the heterozygous allele and about 50% should
have the homozygous allele, that’s 25% dominant and 25% recessive. This is based on the
statistical frequency for the Alu+ allele being 0.5
Method:
Extract cheek cells into a saline solution.
www.cbs.dtu.dk/staff/dave/roanoke
Centrifuge and resuspend the pellet into Chelex
solution
Boil and centrifuge again
Add 5µl supernatant to 44µl reaction mixture and
then ad 1µl DNA polymerase.
Put mixture into the PCR machine and run for 28
Sample results: The image below shows 3 lanes
with fluorescent bands of DNA in each lane. The
markers are of know length and are used to indicate the size of the other bands.
After 45—60min, we can see the separation of the
2 alleles in the heterozygous lane.
Markers
(bp)
PCR
Homozygous
Hetero–
zygous
Denaturing the double helix at 367K
Annealing primer sequences
specific to the relevant gene
766
500
300
DNA polymerase attaches and
starts to replicate from the
150
50
For every cycle,
the amount of
copies of the relevant gene doubles.
It only takes 28
cycles to get
enough copies to
perform electro-
Analysis and conclusion: Fromm looking at electrophoresis samples of all of
the students, it is possible to set up a
distribution for our sample population.
//en.wikipedia.org/wiki/PCR
Electrophoresis:
After PCR there is enough DNA to run electrophoresis with. This can be used to separate the different
alleles according to size.
Distribution of alleles in sample
pupolation.
Dominant
Heterozygous
56%
22%
Recessive
22%
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