Objective
Review DNA replication, knowledge of the process, and
how it occurs.
The concept of PCR
The purpose of DNA and RNA extraction relative to PCR.
The process of PCR
the purpose of a primer
Brief background of history of taq and its purpose in PCR
DNA replication
Replication allows our genetic material to be
passed on to daughter cells.
Replication review
Replication Overview
Origin of replication
Helicase
SSB
RNA primase
Replication Review
Leading strand
Continuous
Lagging strand
Discontinuous
Okazaki fragments
What is PCR???
PCR is a technique used to AMPLIFY a few copies of DNA
exponentially.
Purpose for Amplification
Very small samples can be amplified and
analyzed
DNA found at crime scene investigations
Paternity test
Diagnosis (ex. Viral load in HIV)
How does it work?
You need 5 things:
Template DNA
Primers
Thermal cycling – increase and decrease the
temperature
taq DNA polymerase
dNTPs
What’s a primer?
A primer is a sequence
necessary for replication, which
binds to a target DNA sequence
DNA polymerase requires a
primer to begin synthesizing a
complementary strand
dNTPs
Deoxynucleoside
triphosphates
Triphosphate
Sugar – deoxyribose
Nitrogenous base
A
T
C
G
Thermal cycler
•
Machine used for PCR
amplification
• Raises and lower
temperatures for temperature
sensitive reactions
Steps for PCR
Step 1: Denaturing
Heat 94-98°C
Causes the DNA to separate by disrupting the
hydrogen bonds between strands
Steps for PCR
Step 2: Annealing
Lower temperature to 50-65°C
Allows primer to bind to DNA strand – H bonding
between DNA and primers
One primer binds to 5’ end and one to 3’ end
Steps for PCR
Step 3: Elongation
Temperature depends on polymerase you use
taq DNA polymerase works best at 75-80°C
Polymerase add dNTPs to primer and extends the
strand.
The history of taq
Heating inactivates the DNA polymerase used
before taq was discovered 
Researchers needed a DNA polymerase that
didn’t need replacing after every round of PCR
taq DNA polymerase was discovered in bacteria
called Thermus aquaticus that lived in
underwater hydrothermal vents
taq DNA polymerase!
Overview
This cycle of
heating and cooling
is repeated many
times to amplify the
amount of DNA.
PCR Video
http://www.youtube.com/watch?v=qKlMEdZ7g4Y
Gel
Electrophoresis
Run product on an agarose
gel
Electric current is run through
the gel to separate product
based on size
Ensures the correct product
was amplified
Gel electrophoresis
Ladder – DNA solution of
varying sizes and used to
compare samples
Positive control – a group
where you expect an
outcome
Negative control – a group
where you expect nothing
to happen
Larger fragments are
closer to the top
Questions
What is the purpose of PCR?
Answer
To amplify small amounts of DNA
Question
What are the 3 steps for PCR?
Answer
Questions
If your DNA template strand sequence was
5’AGCTAGGCTAACTGCCGGGC 3’, what would
your primer sequences be if they were 4 bases
long?
Answer
3’TCGA5’
5’GCCC3’
Questions
Why do we use a taq DNA polymerase?
Answer
It can be used at higher temperatures, unlike
previous enzymes which denatured at these
temperature.
Lab today
Today you will be doing PCR on a crime scene
investigation
2 DNA samples were taken from the crime scene
One was skin
The other is saliva
There are 2 suspects: Trisha Jones, and Neil
Burns
Lab today
You will perform PCR on the DNA samples to
amplify it.
The samples will be run on an agarose gel by
electrophoresis to compare the DNA found at
the crime scene to the suspects.
Lab today
There will 2 gels per lab.
2-3 groups will run one gel.
Each gel has 8 wells to run samples.
Lab today
Victim’s DNA
Suspect 1 DNA
Suspect 2 DNA
Saliva
Skin
Negative control
Positive control
DNA ladder
Lab today
Objective: To understand the importance of PCR
and carry out a PCR reaction
Hypothesis: Amplification of the DNA found at
the crime scene will match that of the criminal