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POLYMERASE CHAIN REACTION
What is Polymerase chain
reaction? (PCR)
PCR is a technique that is used to amplify one
sample of DNA thousands of times over to
create a large enough DNA sample for
extensive analysis.
i.e. each time a PCR cycle is performed the total
amount of DNA is doubled.
It is In Vitro amplification of DNA.
What are some uses of PCR?
PCR can be used for many applications
1) Paternity Tests – The child’s tandem repeats
are compared to the mother’s and the
father’s.
2) Detecting mutations- Comparing one
persons DNA to the DNA of the person with
the mutation
The use of PCR in forensics
To create enough DNA from a small sample to
create a DNA profile.
What is needed?
 The DNA fragment to be copied
 DNA polymerase – for PCR it is taken from
bacteria that live in hot springs so it is tolerent to
heat
 Primers – short pieces of DNA that bind to the
start of each end of fragment – also help to
prevent both ends from joining back up
Primers in excess
The annealing reaction is very
efficient because the primers are "in excess" in
the reaction. In a typical PCR reaction, 10,000
molecules of a template may be used, which is
1.6 x 10-20 moles (0.016 attomoles). On the
other hand, 5 picomoles of each primer may
be used (5 x 10-12 moles) -- that is a 3 x 108 fold
excess.
What is needed?
 The DNA fragment to be copied
 DNA polymerase – for PCR it is taken from
bacteria that live in hot springs so it is tolerent to
heat
 Primers – short pieces of DNA that bind to the
start of each end of fragment – also help to
prevent both ends from joining back up
 Nucleotides
 Thermocycler – a computer controlled machine
that varies temperatures precisely for set time
periods
Biorad – PCR song (find on
youtube)
The process of PCR
Step 1: Denature the DNA
“Denature” means to separate the DNA strands into
2 separate strands
This involves heating the DNA sample up to 96 degrees!
The process of PCR
Step 2: Anneal the DNA - “annealing” means to add
In this step 2 primers are added to the 2 separated
DNA strands. ( 1 on each strand)
The temperature is “cooled” to 65 degrees, this
help the primers bind to the DNA.
The primer is an attachment that signals to a
polymerase where to start synthesising (making)
new DNA
The process of PCR
Step 3: “Extension” of DNA- New DNA Created
2 Polymerase molecules attach to the 2 Primers on
the 2 DNA strands and move along the strand.
As they move along they create new
“complementary” DNA.
Temperature goes up to 72 degrees
The process of PCR
Done!
By heating the DNA to separate it into 2 strands
(Denaturation)
Cooling the DNA to add 1 primer to each of the
2 DNA strands (Annealing)
And by Adding 1 polymerase to each strand to
synthesise DNA from where the primer
attached (Extension)
You just doubled the mount of DNA present.
Animation
 http://www.dnalc.org/resources/animations/p
cr.html
Making more DNA
Every time you do a Cycle of PCR you double
the DNA.
You can do this process as many times as
needed to create millions of strands of DNA.
Cycle Number
DNA copies
None
2
1st cycle
4
2nd cycle
8
3rd cycle
16
Now complete these questions
 When you finish you can use the computer
room to run through a simulation
PCR simulation
http://learn.genetics.utah.edu/content/labs/pcr/
PCR simulation
http://learn.genetics.utah.edu/content/labs/pcr/