SY 2014-2015

1. POLYMERASE CHAIN
REACTION

2. WHAT IS PCR?
 PCR is a technique that takes specific
sequence of DNA of small amount and
amplifies it to be used for further testing.
 PCR can make billions of copies of a target
sequence of DNA in a few hours

3. WHY “POLYMERASE”?
 It is called “polymerase” because the only enzyme
used in this reaction is DNA polymerase.
WHY “CHAIN”?
 It is called “chain” because the products of the first
reaction become substrates of the following one,
and so on.

5. SHORT HISTORY OF PCR
 In 1983 Dr. Kary Banks Mullis developed
PCR. He receive a Noble Prize in
Chemistry in 1993 for his invention of the
polymerase chain reaction. It is hailed
as one of the monumental scientific
techniques of the twentieth century.
 In 1985 the first publication of
PCR by Cetus Corporation
appears in Science. Cetus
Corporation was one of the
first biotechnology companies.

6.  1986: Purified Taq polymerase is first used in PCR.
TAQ Polymerase is a DNA Polymerase found in
bacteria that live in thermophilic conditions, such as
hot water springs. It is used to synthesize a new
DNA strand from a template.
SHORT HISTORY OF PCR
 1988: PerkinElmer introduces
the automated thermal cycler.
Also known as
a thermocycler, PCR
machine or DNA amplifier. It is
a laboratory apparatus most
commonly used to amplify
segments of DNA via
the polymerase chain
reaction (PCR).

8. PCR - before the thermocycler
95º C
5 min
35 times
55º C
3 min
72º C
5 min

9. DNA REPLICATION VS. PCR
 PCR 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.
DNA REPLICATION IN CELLS (IN VIVO)
 DNA replication is the copying of DNA
 It typically takes a cell just a few hours to copy all of its
DNA
 DNA replication is semi-conservative (i.e. one strand of
the DNA is used as the template for the growth of a new
DNA strand)
 This process occurs with very few errors (on average
there is one error per 1 billion nucleotides copied)
 More than a dozen enzymes and proteins participate in
DNA replication

10. POLYMERASE CHAIN REACTION
 Polymerase chain reaction enables large amounts
of DNA to be produced from very small samples
(0.1ml)
 There is a repeating cycle of:
separation of double DNA strands
synthesis of a complementary strand for each
 Its purpose is to amplify a lot of double-stranded
DNA molecules (fragments) with same (identical)
size and sequence by enzymatic method and
cycling condition.

11. COMPONENTS
 DNA template - the sample DNA that contains the
target sequence. At the beginning of the reaction, high
temperature is applied to the original double-stranded
DNA molecule to separate the strands from each other.
 DNA polymerase - a type of enzyme that synthesizes
new strands of DNA complementary to the target
sequence. The first and most commonly used of these
enzymes is Taq DNA polymerase (from Thermis
aquaticus), whereas Pfu DNA polymerase
(from Pyrococcus furiosus) is used widely because of its
higher fidelity when copying DNA. Although these
enzymes are subtly different, they both have two
capabilities that make them suitable for PCR: 1) they
can generate new strands of DNA using a DNA template
and primers, and 2) they are heat resistant.

12. COMPONENTS
 Primers - short pieces of single-stranded DNA that
are complementary to the target sequence. The
polymerase begins synthesizing new DNA from the
end of the primer.
 Nucleotides (dNTPs or deoxynucleotide
triphosphates) - single units of the bases A, T, G,
and C, which are essentially "building blocks" for
new DNA strands.

13. BASIC REQUIREMENTS FOR PCR
 DNA sequence of target region must be known.
 Primers - typically 20-30 bases in size.
These can be readily produced by commercial
companies. Can also be prepared using a DNA
synthesizer
 Thermo-stable DNA polymerase - eg Taq
polymerase which is not inactivated by heating to
95C
 DNA thermal cycler - machine which can be
programmed to carry out heating and cooling of
samples over a number of cycles.

14. CONDITION
 Denaturation of ds DNA template
 Annealing of primers
 Extension of ds DNA molecules

15. DENATURATION
 Temperature: 92-94C
 Double stranded DNA melts single stranded
DNA
92C
3’5’
3’ 5’
+
5’3’
5’ 3’

17. ANNEALING
 Temperature: ~50-70C (dependant on the melting
temperature of the expected duplex)
 Primers bind to their complementary sequences
5’3’
5’ 3’
Forward primer Reverse primer

19. EXTENSION
 Temperature: ~72C
 Time: 0.5-3min
 DNA polymerase binds to the annealed primers and
extends DNA at the 3’ end of the chain
Taq
5’
3’
Taq5’

21. CYCLING

22. PRODUCTS OF EXTENSION
3’5’
3’ 5’
3’5’
3’ 5’
Taq
Taq

23. OVERALL PRINCIPLE OF PCR
 DNA – 1 copy
Known sequence Sequence of interest Known sequence
 PCR

24. ADVANTAGES
 Much faster than using vectors
 Only a little bit of target DNA is needed

25. DISADVANTAGES
 Are to synthesize primers, we need to know the
sequence flanking the DNA segment of interest
 Only applies to short DNA fragments, mostly less
than 5 kb

26. WHAT IS IT USED FOR?
 Medical and biological research
 Cloning
 Diagnosis of hereditary diseases
 Identification of fingerprints
 Diagnosis of infectious diseases