Recombinant DNA
Technology………..
BTEC3301
Polymerase Chain Reaction (PCR)
Introduction
•
The Polymerase Chain Reaction
(PCR) provides an extremely
sensitive means of amplifying small
quantities of DNA.
•
Kary Mullins won the Nobel Prize
in 1993 for development of this
novel technique.
Polymerase Chain Reaction (PCR)
Introduction
•
The technique was made possible by
the discovery of Taq polymerase,
the DNA polymerase that is used by
the bacterium Thermus auquaticus ,
discovered in hot springs.
Polymerase Chain Reaction (PCR)
Introduction
•
The use of thermostable DNA
polymerases was an important
breakthrough since this enzyme
does not denature at the
temperatures( 950C) used to cause
denaturation of the DNA.
Polymerase Chain Reaction (PCR)
Introduction
•
The most well-known of these
thermostable DNA polymerases is
Taq.
•
This enzyme has a molecular size of
94kD and an optimum reaction
temperature of 75-80oC.
Polymerase Chain Reaction (PCR)
Introduction

Amplification means making
multiple identical copies
(replicates) of a DNA sequence.

PCR method of DNA
amplification has proved very
important in recombinant DNA
technology and is used in a
range of applications in
medicine and forensic science .
Polymerase Chain Reaction (PCR)
What is PCR?
•
It is used to amplify a specific DNA
(target) sequence lying between
known positions (flanks) on a
double-stranded DNA molecule.
•
The amplification process is
mediated by oligonucleotide primers
that, typically, are 20-30
nucleotides long.
Polymerase Chain Reaction (PCR)
What is PCR?
•
The primers are single-stranded
DNA.
•
Primers anneal to the flanking
regions by complementary-base
pairing (G=C and A=T) using
hydrogen bonding.
Polymerase Chain Reaction (PCR)
•
The amplified product is known as
an amplicon.
•
Generally, PCR amplifies smallish
DNA targets 100-1000 base pairs
(bp) long.
Polymerase Chain Reaction (PCR)
Material required in PCR process:
•
Thermal cycler (PCR machine,
available in different specificity and
models).
Polymerase Chain Reaction (PCR)
:
 PCR mix for a reaction has the
following:
sample DNA with a target sequence
 thermostable DNA polymerase (Taq is
commonly used)
 two oligonucleotide primers which are
complementary to the sequence
flanking the target sequence.

Polymerase Chain Reaction (PCR)

deoxynucleotide triphosphates
(dNTPs)

reaction buffer containing
magnesium ions and other
components. Homework:Function
of Magnesium for Quiz!!!
Polymerase Chain Reaction (PCR)
Steps in PCR reactions:
•
1.
Heat denaturation

A DNA molecule carrying a target
sequence is denatured by heat at
90-95oC. The two strands
separate due to breakage of the
hydrogen bonds holding them
together.
Polymerase Chain Reaction (PCR)
•
2. Primer annealing

Primers are added to the
dissociated target strands and
incubated together first at a
temperature that allows the primers
to hybridize to the target strands
(usually somewhere between 40
and 55 oC).
Polymerase Chain Reaction (PCR)
•
3. Primer extension

DNA polymerase is then added and
complementary strands are
synthesized at a temperature of 6075oC. The polymerase causes
synthesis of new material in the 5'
to 3' direction away from each of
the primers.
Polymerase Chain Reaction (PCR)

This allows a first round of synthesis
to occur on each of the DNA
templates.

Following primer extension, the
mixture is heated (again at 90-95oC)
to denature the molecules and
separate the strands and the cycle
repeated
Polymerase Chain Reaction (PCR)

Each new strand then acts as a
template for the next cycle of
synthesis.

Amplification (replication) proceeds
at an exponential (logarithmic) rate
(amount of DNA produced doubles at
each cycle).
Polymerase Chain Reaction (PCR)

A thermal cycler (a machine that
automatically changes the
temperature at the correct time for
each of the stages and can be
programmed to carry out a set
number of cycles) is used for a PCR
reaction.
Polymerase Chain Reaction (PCR)

The amplified product can be
detected using gel electrophoresis to
view the band containing DNA
fragments of a particular size
containing the gene of interest in the
original starter DNA sample.
Diagrammatic steps in PCR process
(Read summary):
•
Three major steps in PCR:
1. Template denaturation
2. Primer annealing
3. Primer extension
Home work for next class!!
Summarize the steps in PCR process
• The oligonucleotides serve as primers for
DNA polymerase and the denatured strands
of the large DNA fragment serves as the
template.
•
This results in the synthesis of new DNA
strands which are complementary to the
parent template strands.
• After each cycle the newly synthesized DNA
strands can serve as templates in the next
cycle.
Polymerase Chain Reaction (PCR)
Diagrammatic steps in PCR process
• PCR amplification is achieved by using
oligonucleotide primers.
Polymerase Chain Reaction (PCR)
Diagrammatic steps in PCR process
•
Fig. 3.8 The Polymerase Chain
Reaction
Go to Animation courtesy of the
following websites:
References
http://faculty.plattsburgh.edu/donald.slish/PCRmov.html (Animation)
http://www.accessexcellence.org/RC/AB/IE/PCR_Xeroxing_DNA.html
 http://www.people.virginia.edu/~rjh9u/pcranim.html ( PCR Animation)
 http://www.escience.ws/b572/L3/L3.htm (PCR Animation)
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http://homepages.strath.ac.uk/~dfs99109/BB211/RecombDNAtechlect4.html
http://en.wikipedia.org/wiki/Polymerase_chain_reaction
http://www.escience.ws/b572/L3/L3.htm
 http://allserv.rug.ac.be/~avierstr/principles/pcrani.html
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