Transcript Human Alu Insertion Polymorphism Experiment

Detection of the human
Mitochondrial DNA
A Polymerase Chain Reaction
Experiment
Polymerase Chain Reaction: another
method of DNA amplification
Basis of PCR

Create conditions “in vitro” for DNA replication.
Requirements for replication
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DNA template
Primers
Taq DNA Polymerase**
Nucleotides
MgCl++
Temperature cycles plays a key role:
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Unwinding DNA
Annealing
Extension
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98C
45-60C
72C
http://www.dnalc.org/Shockwave/pcranwhole.html
Primers determine
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The sequence of DNA that will be amplified.
Basis for sequence specific amplification
•Two primers are used to bracket the area you want to amplify.
•Primers are single stranded synthetic sequences of DNA normally 20-30 bp.
•One primer is complementary to the beginning of the target gene on one
strand while the other primer is complementary to end of the target gene on
the complementary strand.
Summary:
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Unwinding DNA
Annealing
Extension
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98C
45-60C
72C
http://www.dnalc.org/Shockwave/pcranwhole.html
The experiment is divided into 2 parts
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Isolate human DNA from cheek cells
The DNA will be amplified using PCR
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The DNA will be analyzed using gel electrophoresis
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Mitochondrial DNA
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Circular
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16,569 bp
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37 genes
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Encodes 13 polypeptides
Mitochondria
DNA
Proteins of electron transport system
ISOLATION OF DNA AND PCR PROTOCOL
FLOW CHART
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Isolation cheek cell DNA
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Amplify using PCR
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Identify PCR-DNA product by gel electrophoresis
Each person should obtain
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Cotton swabs
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A test tube with 2 mls of buffer
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A screw top microfuge tube
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2 transfer pipets
Isolation of DNA
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Cheek Cells
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Obtain cells by swabbing the inside of
the mouth with a cotton tipped
applicator.
Next…
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Place cotton head in 2 ml of PBS (in conical
tube)
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Swirl vigorously for 1 min. to dislodge cells
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Press the cotton head against the walls to
squeeze out as much liquid as possible
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Use new applicator and repeat the above steps.
Next…
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Transfer 2 ml to screw top tube.
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Spin at 5000 rpm for 5 min. Be sure you
have pellet. If not repeat swabbing.
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Remove supernantant but SAVE PELLET
(these are your cells!)
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Add 100 ul of chelating agent to sample
Next…
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Re-suspend the pellet in 100 ul of
chelator solution: MAKE SURE
PELLET IS RESUSPENDED!
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Place screw top microfuge tube in
boiling waterbath for 10 minutes to
break (lyse) cells.
Next:
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After 10 min boiling allow tube to
cool (2 minutes and then spin
microfuge tube for 2 minutes (5000
rpm)
Next:
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Obtain a PCR tube with “PCR bead”
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Add 20 ul primers to bead and 5 ul of your
supernatant and gently mix.
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Label your PCR tube your assigned seat
number
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Place in PCR!
Summary of PCR tube
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To PCR tube containing “bead” add:
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20.0 ul of primer solution
5.0 ul of cheek cell DNA
Mt DNA : PCR reaction 30 cycles at:
Initial denaturation at 94C for 4 minutes
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94C
55C
72C
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60 seconds
60 seconds
60 seconds
Final Extension: 72C for 5 minutes
EXPECTED FRAGMENT SIZES: 921 and 672
The gel electrophoresis
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Analysis of your results will be carried
and a photo taken.
After PCR
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Gel electrophoresis
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Warm samples (including ladder) 2 minutes at 50 C
Load 25 ul of the sample
Load 25 ul of ladder