What is PCR?
PCR is a technique that takes
a specific sequence of DNA of
small amounts and amplifies
it to be used for further
testing.
Kerry Mullis's
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History of PCR
On April 25, 1953 James D. Watson and Francis Crick published "a radically different
structure" for DNA thereby founding the field of molecular genetics
Starting in the mid 1950s, Arthur Kornberg began to study the mechanism of DNA
replication By 1957 he has identified the first DNA polymerase
In 1969 Thomas D. Brock reported the isolation of a new species of bacterium from a hot
spring in Yellowstone National Park. Thermus aquaticus (Taq),
Taq polymerase isolated by Chien et al. in 1976 became a standard source of enzymes able to
withstand higher temperatures than those from E. Coli
In 1970 Klenow reported a modified version of DNA Polymerase I from E. coli. Treatment
with a protease removed the 'forward' nuclease activity of this enzyme. The overall activity of
the resulting Klenow fragment is therefore biased towards the synthesis of DNA, rather than
its degradation
Mullis, Kary B. (April 1990). "The Unusual Origin of the Polymerase Chain
Reaction". Scientific American. 262 (4): 56–65.
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DNA Amplification
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PCR
PCR is a means to amplify a particular piece of DNA .
Amplify= making numerous copies of a segment of DNA.
PCR can make billions of copies of a target sequence of DNA in short time.
It is a laboratory version of DNA Replication in cells.
The laboratory version is commonly called “in vitro” since it occurs in a test
tube while “in vivo” signifies occurring in a living cell.
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Amplification of specific target sequence
PCR does not copy all of the DNA in the sample. It copies only a very specific sequence of
genetic code from a template DNA, targeted by PCR primers.
It does require the knowledge of some DNA sequence information which flanks the fragment
of DNA to be amplified (target DNA).
From this information two synthetic oligonucleotide primers may be chemically synthesised
each complementary to a stretch of DNA to the 3’ side of the target DNA, one
oligonucleotide for each of the two DNA strands (DNA polymerase can add a nucleotide
only onto a preexisting 3'-OH group)
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Amplification of specific target sequence
Why do we require two primers?
In a PCR reaction you need two primers to
amplify the target sequence:
One called: Forward primer, which have the
same sequence of forward DNA strand and
bind to the complementary reverse strand.
The second called: Reverse primer, which
have the same sequence of reverse DNA strand
and bind to the complementary forward strand.
Primer designing
Criteria for primer designing
Length of 18-24 bases
40-60% G/C content
Start and end with 1-2 G/C pairs
Melting temperature (Tm) of 50-60°C
Primer pairs should have a Tm within 5°C of each other
Primer pairs should not have complementary regions
The “Reaction” Components
Target DNA - contains the sequence to be amplified
Pair of Primers - oligonucleotides that define the
sequence to be amplified.
dNTPs - deoxynucleotidetriphosphates: DNA building
blocks.
Thermostable DNA Polymerase - enzyme that catalyzes
the reaction
Mg++ ions - cofactor of the enzyme
Buffer solution – maintains pH and ionic strength of the
reaction solution suitable for the activity of the enzyme
PCR Mixture
The Reaction
PCR tube THERMOCYCLER
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The PCR Cycle
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Denaturation
The double-stranded template DNA is denatured by heating, typically
to 95°C, to separate the double stranded DNA (why?).
Breaking the _______ bonds.
N_JXUMKT3HUTJY
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Annealing
The reaction is rapidly cooled to the primer annealing temperature (50-65
°C) to allow the oligonucleotide primers to hybridize to single stranded
template.
Primer will anneal only to sequences that are complementary to them (target
sequence).
Extension
The reaction is heated to a temperature depends on the DNA
polymerase used.
Commonly a temperature of 72°C is used with this enzyme.
At this step the DNA polymerase synthesizes a new DNA
strand complementary to the DNA template.
Program in thermos cycler
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PCR Reaction
At the end of the PCR reaction,
the specific sequence will be
accumulated in billions of
copies (amplicons).
In only 20 cycles, PCR can
product about a million (220)
copies of the target.
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Taq DNA Polymerase
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PCR Advantages and application
Simplicity, easier methodology, sensitive, extensively validated standard
operating procedure and availability of reagents and equipment.
Applications
Genotyping.
RT-PCR.
Cloning.
Mutation detection.
Sequencing.
Microarrays.
Forensics.
Paternity testing
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Types of PCR
Nested PCR
RT-PCR
Inverse PCR
Anchored PCR
RACE
Touchdown PCR
Hot start PCR
Multiplex PCR
q-PCR
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Thank you
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