1. POLYMERASE CHAIN
REACTION
29-07-2011 Presented by: Dr.Praveenkumar Doddamani
Department of Microbiology M.R.Medical College,Gulbarga.

2.  INTRODUCTION
 HISTORY
 WHT IS PCR?
 PRINCIPLE OF PCR
 EQUIPMENTS & ELEMENTS OF PCR
 STEPS OF PCR CYCLE
 VARIATIONS OF PCR
 ADVANTAGES OF PCR
 LIMITATIONS OF PCR
 APPLICATIONS OF PCR

3. INTRODUCTION
 PCR is powerful Method of In-vitro DNA
synthesis.
 PCR has revolutionized molecular biology and
is used in virtually every area of natural
science and medicine .
 This technique has cut across the boundaries
separating basic and applied
research, commercial technology and

4. History
 1983—Kary Mullis, a scientist working for
the Cetus Corporation was driving along
US Route 101 in northern California when
he came up with the idea for the
polymerase chain reaction
 1985—the polymerase chain reaction was
introduced to the scientific community at a
conference in October.

5. Polymerase Chain Reaction
Methodology: A Mile stone in Medical
History
 He had the idea to use a
pair of primers to bracket
the desired DNA
sequence and to copy it
using DNA polymerase, a
technique which would
allow a small strand of
DNA to be copied
almost an infinite
number of times. Cetus
took Mullis off his usual
projects to concentrate on
PCR full-time

6.  Cetus rewarded Kary Mullis with a $10,000
bonus for his invention
 Later, during a corporate
reorganization, Cetus sold the patent for the
PCR process to a pharmaceutical company
Hoffmann-LaRoche for $300 million.
 1993 Kary Mullis got NOBEL PRIZE for
chemistry.

7. Dr. Kary Mullis, wins Nobel Prize in
1993
 Kary received a Nobel
Prize in chemistry in
1993, for his invention of
the polymerase chain
reaction (PCR). The
process, which Kary
Mullis conceptualized in
1983, is hailed as one of
the monumental
scientific techniques of
the twentieth century.

8. What is PCR?
PCR is an exponentially
progressing synthesis of the
defined target DNA sequences in
vitro.

9. 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.

10. The ―Reaction‖ Components
1) Target DNA - contains the sequence to be amplified.
2) Pair of Primers - oligonucleotides that define the sequ
to be amplified.
3) dNTPs - deoxynucleotidetriphosphates: DNA building
4) Thermostable DNA Polymerase - enzyme
that catalyzes the reaction
5) Mg++ ions - cofactor of the enzyme
6) Buffer solution – maintains pH and ionic
strength of the reaction solution suitable for
the activity of the enzyme

12. Principle of
PCR
 The principle of PCR is rather simple and involves
enzymatic amplification of a DNA fragment
flanked by two oligonucleotides (primers)
hybridized to opposite strands of the template
with the 3’ends facing each other.
 DNA polymerase synthesizes new DNA starting
from the 3’ end of each primer.
 Repeated cycles of heat denaturation of the
template, annealing of the primers and extension
of the annealed primers by DNA polymerase
results in amplification of the DNA fragment. The
extension product of each primer can serve as

16. The Reaction
PCR tube THERMOCYCLER

17. Elements of standard PCR
reaction
 Components of a standard PCR reaction are:
 Thermostable DNA polymerase,
 DNA template,
 primers,
 dNTP substrate,
 MgCl2 buffer and salt.
 In addition, PCR reactions frequently include
compounds that stabilize the enzyme and
reagents that help DNA dissociation or primer
annealing.

18.  DNA polymerase
Most common thermostable DNA polymerase is
Taq polymerase. ( Thermus aquaticus)
Properties that make it less than ideal:
 First, the enzyme has very high error rates
due to the lack of 3’to 5’exonuclease activity.
 Second, the enzyme adds nucleotides to 3’
ends in a template-independent
manner, making the amplification product
difficult to clone.
 Third, the enzyme is quite expensive.

19.  Variety of thermostable DNA polymerases
from thermophilic or hyperthermophilic
bacteria.
 Standard PCR reactions (e.g., Tli, Pvo) ,
 Sequencing (e.g., Pvo, AmpliTherm),
 Long PCR reaction mixtures(e.g.Tth).
 A used concentration is 2.0 units/100 µl
reaction.

20.  DNA template
 One of the most important features of PCR is
that it can be performed with very small
quantities of relatively impure DNA. Even
degraded DNA can be successfully amplified.
 Therefore a number of simple and rapid
protocols to purify DNA for PCR have been
developed.
 Numbers of contaminants efficiency of
amplification. Ex: urea, SDS, sodium acetate
and some components eluted from Agarose can
interfere with PCR.
 Most of these impurities can be removed by
washing with 70% ethanol or by reprecipitation
of DNA in the presence of ammonium acetate.

21. Primers: guidelines
 Optimal primer set should hybridize to the template
efficiently with negligible hybridization to other
sequences of the sample. primers should be at least
20 to 25 bases in length. However, RAPD analysis
primers are usually 8 to 10 bases in length.
 Primers, if possible, should have GC content similar
to that of the target & both primers similar GC.
 Primers should not have sequences with significant
secondary structure. No simple repeats or
palindromic sequences.
 Primer pairs should not contain complementary
sequences to each other. primers with less 3’ overlaps

22. Primers That Form Hairpins
 Primers can have self-annealing regions within each
primer (i.e. hairpin and fold back loops)
 A primer may be self-complementary and be able to
fold into a hairpin:
5´-GTTGACTTGATA
||||| T
3´-GAACTCT
 The 3´ end of the primer is base-paired,
preventing it annealing to the target DNA.

23. Substrate
 Conc.of each of dNTP in PCR should not exceed
200 µM.
 200 µM dNTP’s→12.5 µg of DNA when half of
the nucleotides are incorporated.
 All 4 dNTPs should be used at equivalent
concentrations , This will minimize the error rate
of the enzyme.
 An excess of nucleotides inhibits enzyme activity
and can contribute to the appearance of false
products.

24. MgCl2 Concentration
 Mg ion is a required co-factor for all DNA
polymerases.
 magnesium ion concentration may affect the
following:
 Primer annealing.
 Temperature of strand dissociation for
template and product.
 Product specificity.
 Formation of primer-dimer artifacts and
enzyme fidelity.
 Many templates require optimization of
magnesium ion concentration for efficient,

25. Buffer
 A standard buffer for PCR is 10 to 50 mM Tris-
HCL. The optimum pH is between 8-9 for most
thermophilic polymerases. Since the Δ pKa for
Tris is high(-0.031/ C), the true pH of the
reaction mixture during a typical thermal cycle
varies considerably (approximately 1 to 1.5 pH
units).

26. SALTS : in Buffer
 The salt used in most reactions is K /Na, added
to facilitate correct primer annealing. For Taq
polymerase, the conc.is 50 mM.
 Other components stabilize the enzyme are:
gelatin, bovine serum albumin or nonionic
detergent(Tween 20 or Triton X 100). Most
protocols work well without.
 When using DNA template(high GC
content), the reaction mixture also includes
reagents to lower the Tm (melting temp) of the
template. Among these are DMSO, acetamide
or glycerol.

27. Thermal cycling profile
Standard PCR consists of three steps;
 Denaturation, Annealing and Extension.
 These steps are repeated or cycled 25-30
times.
 Most protocols also include a single
Denaturation step (Initial Denaturation) before
cycling begins and single long extension step
(final extension) at the end.

30. Steps in PCR
 Denaturation 93 to 95 C
1min
 Annealing 50 to 55 C
45sec
 Elongation 70 to 75 C

31. Denaturation of DNA
Denaturation is the first step
in PCR, in which
the DNA strands are
separated by heating to
95°C.
The Hydrogen bonds
between the two strands
breaks down and the two
strands separates.

32. Annealing
Annealing is the process of
allowing two
sequences of DNA to form
hydrogen bonds.
The annealing of the target
sequences and
primers is done by cooling
the DNA to 55°C.
Time taken to anneal is 45
seconds

33. Taq polymerase binds ….
Taq polymerase binds to the
template DNA
and starts adding
nucleotides that are
complementary to the first
strand.
This happens at 72°C as it
is the optimum
temperature for Taq
Polymerase.

34. Elongation step
In this step,
 DNA polymerase synthesizes a new DNA strand by
extending the 3’ end of the primers.
 Time of the elongation depends on the length of the
sequence to be amplified. Since Taq polymerase can
add 60-100 bases per second under optimal conditions,
synthesis of a 1Kbp fragment should require a little less
than 20 seconds.
 most protocols recommend 60 seconds per 1 Kbp DNA
to account for time needed to reach the correct
temperature and to compensate for other unknown
factors that can affect reaction rate.
 The shortest possible time should be used to preserve

35. Ta
q

38. The target product is made
only in the third cycle
5’ 3’
3’ 5’
Cycle 1 5’ 3’
3’ 5’
3’
5’
3’ 5’
Cycle 2 5’ 3’
3’ 5’
3’ 5’
Cycle 3 5’ 3’
3’ 5’
5’ 3’

40. After 30
cycles this
becomes
one billion!
230

41. DNA copies vs Cycle number
2500000
2000000
DNA copies
1500000
1000000
500000
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Cycle number

42. The PCR process can be divided into
three stages
 Exponential amplification: At every cycle, the
amount of product is doubled (assuming 100%
reaction efficiency). The reaction is very sensitive:
only minute quantities of DNA need to be present.
 Leveling off stage: The reaction slows as the DNA
polymerase loses activity and as consumption of
reagents such as dNTPs and primers causes
them to become limiting.
 Plateau: No more product accumulates due to
exhaustion of reagents and enzyme

44. PCR Cycles Review
 Denaturation: 94 - 95 C
 Primer Annealing: 55 - 65 C
 Elongation of DNA: 72
 Number of Cycles: 25-40
 No target products are made until the
third cycle.
 At 30 cycles there are 1,073,741,764
target copies (~1 109).

45. Variations of the PCR
 Colony PCR
 Nested PCR
 Multiplex PCR
 AFLP PCR
 Hot Start PCR
 In Situ PCR
 Inverse PCR
 Asymmetric PCR
 Long PCR
 Long Accurate PCR
 Reverse Transcriptase PCR
 Allele specific PCR
 Real time PCR

46. Advantages of PCR
 Speed
 Ease of use
 Sensitivity
 Robustness

47. Limitations of PCR
 Need for target DNA sequence information
Primer Designing for unexplored ones.
Boundary regions of DNA to be amplified must be known.
 Infidelity of DNA replication.
Taq Pol – no Proof reading mech – Error 40% after 20
cycles
 Short size and limiting amounts of PCR product
Up to 5kb can be easily amplified .
Up to 40kb can be amplified with some modifications.
Cannot amplify gene >100kb
Cannot be used in genome sequencing projects.

48. Applications of PCR
 Medical applications
 Infectious disease applications
 Forensic applications
 Research applications

49. Applications of PCR
Basic Research Applied Research
• Mutation screening • Genetic matching
• Drug discovery • Detection of pathogens
• Classification of organisms • Pre-natal diagnosis
• Genotyping • DNA fingerprinting
• Molecular Archaeology • Gene therapy
• Molecular Epidemiology
• Molecular Ecology
• Bioinformatics
• Genomic cloning
• Site-directed mutagenesis
• Gene expression studies

50. Applications of PCR
Molecular Identification
Sequencing Genetic Engineering
• Molecular Archaeology • Bioinformatics • Site-directed mutagenesis
• Molecular Epidemiology • Genomic cloning • Gene expression studies
• Molecular Ecology • Human Genome Project
• DNA fingerprinting
• Classification of organisms
• Genotyping
• Pre-natal diagnosis
• Mutation screening
• Drug discovery
• Genetic matching
• Detection of pathogens

51. MICROBIOLOGICAL APPLICATION OF PCR:
•A
•B
•C
•D
•E

52. Conclusion
The speed and ease of
use, sensitivity, specificity and
robustness of PCR has revolutionised
molecular biology and made PCR the most
widely used and powerful technique with great
spectrum of research and diagnostic
applications.

54. Colony PCR
Colony PCR- the screening of bacterial (E.Coli) or yeast clones for
correct ligation or plasmid products.
Pick a bacterial colony with an autoclaved toothpick, swirl it into 25 μl
of TE autoclaved dH2O in an microfuge tube.
Heat the mix in a boiling water bath (90-100C) for 2 minutes
Spin sample for 2 minutes high speed in centrifuge.
Transfer 20 μl of the supernatant into a new microfuge tube
Take 1-2 μl of the supernatant as template in a 25 μl PCR standard
PCR reaction.

55. Hot Start PCR
 This is a technique that reduces non-specific amplification
during the initial set up stages of the PCR
 The technique may be performed manually by heating the
reaction components to the melting temperature (e.g., 95°C)
before adding the polymerase
 Specialized enzyme systems have been developed that inhibit
the polymerase's activity at ambient temperature, either by the
binding of an antibody or by the presence of covalently
bound inhibitors that only dissociate after a high-temperature
activation step
 DNA Polymerase- Eubacterial type I DNA polymerase, Pfu
 These thermophilic DNA polymerases show a very small
polymerase activity at room temperature.

56. Asymmetric PCR
 Asymmetric PCR is used to preferentially amplify
one strand of the original DNA more than the other.
 It finds use in some types of sequencing and
hybridization probing where having only one of the
two complementary stands is ideal.
 PCR is carried out as usual, but with a great excess
of one primers for the chosen strand.

57. Nested PCR
Two pairs (instead of one pair) of PCR primers
are used to amplify a fragment.
 First pair -amplify a fragment similar to a standard
PCR. Second pair of primers-nested primers (as
they lie / are nested within the first fragment) bind
inside the first PCR product fragment to allow
amplification of a second PCR product which is
shorter than the first one.
 Advantage- Very low probability of nonspecific
amplification

59. AFLP PCR
AFLP is a highly sensitive PCR-based method
for detecting polymorphisms in DNA. AFLP
can be also used for genotyping individuals
for a large number of loci
•Genomic DNA is digested with one or more
restriction enzymes. tetracutter (MseI) and a
hexacutter (EcoRI).
•Ligation of linkers to all restriction fragments.
• Pre-selective PCR is performed using primers
which match the linkers and restriction site
specific sequences.
•Electrophoretic separation and amplicons on a
gel matrix, followed by visualisation of the band
pattern.

60. Inverse PCR
 Inverse PCR (Ochman et al., 1988) uses standard PCR
(polymerase chain reaction)- primers oriented in the
reverse direction of the usual orientation.
 The template for the reverse primers is a restriction
fragment that has been selfligated
 Inverse PCR functions to clone sequences flanking a known
sequence. Flanking DNA sequences are digested and then
ligated to generate circular DNA.
Applications
 Amplification and identification of sequences flanking
transposable elements, and the identification of genomic
inserts.

62. Multiplex PCR
 Multiplex PCR is a variant of PCR which enabling
simultaneous amplification of many targets of
interest in one reaction by using more than one pair
of primers.

63. In Situ PCR
 In Situ PCR (ISH) is a polymerase chain reaction that
actually takes place inside the cell on a slide. In situ
PCR amplification can be performed on fixed tissue or
cells.
 Applies the methodology of hybridization of the nucleic
acids.
 Allows identification of cellular markers
 Limited to detection of non-genomic material such as
RNA, genes or genomes

64. In Situ PCR

65. Long PCR
 Extended or longer than standard PCR, meaning over 5
kilobases (frequently over 10 kb).
 Long PCR is useful only if it is accurate. Thus, special
mixtures of proficient polymerases along with accurate
polymerases such as Pfu are often mixed together.
 Application- to clone large genes not possible with
conventional PCR.

66. Reverse Transcriptase
PCR
 Based on the process of reverse transcription, which
reverse transcribes RNA into DNA and was initially isolated
from retroviruses.
 First step of RT-PCR - "first strand reaction―-Synthesis of
cDNA using oligo dT primers (37°C) 1 hr.
 ―Second strand reaction―-Digestion of cDNA:RNA hybrid
(RNaseH)-Standard PCR with DNA oligo primers.
 Allows the detection of even rare or low copy mRNA
sequences by amplifying its complementary DNA.

68. Allele-specific PCR
 Used for identify of SNPs.
 It requires prior knowledge of a DNA
sequence, including differences between alleles.
 Uses primers whose 3' ends encompass the SNP
 PCR amplification under stringent conditions is
much less efficient in the presence of a mismatch
between template and primer
 Successful amplification with an SNP-specific
primer signals presence of the specific SNP in a
sequence

69. What is Real Time PCR?
Real Time PCR is a technique in which
fluoroprobes bind to specific target regions of
amplicons to produce fluorescence during PCR.
The fluorescence, measured in Real Time, is
detected in a PCR cycler with an inbuilt filter
flurometer.