Lab 8:
PCR
(Polymerase Chain Reaction)
By Kristi Schramm
Gabrielino HS
PCR – Polymerase Chain
Reaction has many applications
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PCR is commonly used to produce many copies
of a selected gene segment or locus of DNA.
In criminal forensics, PCR is used to amplify
DNA evidence from small samples that may
have been left at a crime scene.
PCR can be used to amplify DNA for genetic
disease screening
Lab 8: Obtaining DNA Sample
1)
2)
3)
4)
Add cheek cells to Chelex
Boil (lyse cells and destroy nuclease)
Centrifuge
Extract DNA sample
Lab 8:
PCR
(Polymerase Chain Reaction)
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The locus we will amplify is located in the
tissue Plasminogen Activator (tPA) gene.
This gene is on chromosome 8.
The gene codes for a protein that is
involved with dissolving blood clots.
tPA is a protein given to heart attack
victims to reduce the incidence of strokes.
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The region we will be amplifying is
located in an intron (non-translated
region), of the tPA gene.
Quick Review on Exons and
Introns
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The intron that we will be
targeting for amplification is
dimorphic, which means the locus
has two forms.
one form carries a 300 bp DNA
fragment known as an Alu
element
the second form of the locus
does not carry this fragment.
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The diagram
indicates the
intron we will be
targeting for
PCR.
Two Possibilities:
100 bp sequence
400bp sequence
What are Alu elements?
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Alu elements are short, around
300 bp, DNA fragments that are
distributed throughout our
genome.
Estimated that we may carry
over 1,000,000 copies of this
fragment.
The PCR
Reaction
How does
it work?
Heat (94oC) to denature DNA
strands
Cool (56oC) to anneal primers to
template
Warm (72oC) to activate Taq
polymerase, which extends
primers and replicates DNA
Repeat 40 cycles
PCR
1) 94 C: Denature DNA
2) 56 C: Anneal Primers to Template
3) 72 C: Activates Taq Polymerase
• Repeats 31 times
What is needed for PCR?
The PCR
Reaction
What do
you
need?
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Template - the DNA to be amplified
Primers - 2 short specific pieces of DNA
whose sequence flanks the target
sequence
 Forward
 Reverse
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Nucleotides - dATP, dCTP, dGTP, dTTP
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Magnesium chloride - enzyme cofactor
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Buffer - maintains pH & contains salt
Taq DNA polymerase – thermophillic
enzyme from hot springs (Thermus
aquaticus)
What do we use?
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Reagents and supplies
Equipment
and supplies
Genomic DNA sample (5 µL)
P-20 pipette and tips
Master mix I (10 µL/reaction)
Thermal cycler
2.5 µL 10x PCR buffer w/o MgCl2
0.5 µL dNTP’s (10 mM)
2.5 µL Forward primer (4pM/ µL)
2.5 µL Reverse primer (4pM/ µL)
0.15 µL Taq polymerase
1.85 µL ddH2O
Master mix II (10 µL/reaction)
0.75 µL MgCl2 (50 mM)
9.25 µL ddH2O
Expected Results of PCR
1.
2.
3.
4.
Marker
Homozygous Alu +
Homozygous Alu –
Heterozygous
Expected Results
Huntington Disease
Trinucleotide repeats
(CAGCAGCAG…)
 Over 40 of these repeats causes the
disease
 PCR can be used to identify this
disease
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The Alu element maybe a part of the
DNA coding for an RNA molecule that
aids in the secretion of newly formed
polypeptides from the cell.
it has little if any effect on protein
function unless it happens to become
inserted into an exon or coding region