PCR- DNA AMPLIFICATION

2. DNA amplification
In vivo In vitro
(Cloning) (PCR)

3. Method first proposed by H. G. Khorana & colleagues in
1970’s.
 15 years later the idea was independently conceived by
Karry Mullis in 1983.
 Used the Klenow fragment of E. coli DNA polymerase to
describe the in-vitro amplification of genes.
 Saiki et al in 1988 used the thermostable DNA
polymerase from Thermus aquaticus and greatly
increased the efficiency of PCR.
 In 1989, Science magazine selected PCR as the major
scientific development and Taq DNA polymerase as the
molecule of the year.
 Karry Mullis was awarded the Noble prize for chemistry
in 1993.

4.  An in vitro method for enzymatically synthesizing
defined sequences of DNA
 The technique has been a revolution in molecular
biology and now is so pervasive that it is difficult to
imagine life without it.
 The problem of insufficient DNA is no longer a
problem in molecular biology research or DNA-
based diagnostics.

5.  It’sWhy Polymerase?

a means of selectively amplifying a particular
segment of DNA.
 It is called “polymerase” because the only
enzyme used in this reaction is DNA
 The segment may represent a small part of a large
polymerase.
and complex mixture of DNAs:
 Why Chain?
e.g. a specific exon of a human gene.
 It is called “chain” because the products of the
first reaction become substrates of the
 It can be thought ofandaso on.
following one, as molecular photocopier.
 The “Reaction” components

6. Essential components required:
 Template DNA
 A thermostable DNA polymerase
 A pair of synthetic oligonucleotide primers.
 Divalent cations (Mg 2+ )
 dNTPs
 Buffer to maintain pH

7.  Various types
• Single or double stranded DNA
• Genomic, cloned, bacterial, viral
• RNA/cDNA
 Closed circular DNA templates are amplified
slightly less efficiently than linear ones.
 Amplification depends on the number of copies
of the target DNA seeded into the reaction.

8.  Needs a pre-existing DNA to duplicate
◦ Cannot assemble a new strand from
components
◦ Called template DNA
 Can only extend an existing piece of DNA
◦ Called primers
5’ 3’
3’ 5’

9.  PCR uses the enzyme DNA polymerase that directs the
synthesis of DNA from deoxynucleotide substrates on a
single-stranded DNA template
 A wide range of thermostable polymerases are available,
which vary in their fidelity, efficiency and ability to
synthesize large DNA products.
 Taq polymerase isolated from Thermus aquaticus is the
first isolated and best known enzyme.

10. Taq polymerase
Source Thermus aquaticus
Activity 5’ – 3’ polymerase activity, but
lacks 3’ – 5’ exonuclease activity
(no proofreading)
Stability Half life of <5 min at 100 C, but
retains activity up to 40 min at 95°C
Error rate 2 x 10-4 errors / base
Fidelity low
When greater fidelity is required, other thermostable
enzymes may have significant advantages.

11. Enzyme Source Optimum Fidelity Proofreading
temp. C
rTth T. thermophilus 75-80 Low none
Pfu Pyrococcus 72-78 High Yes
furiosus
Pwo P. woesei 60-65 High Yes
Deep Pyrococcus 70-80 High Yes
Vent strain GB-D
Cocktails of different enzymes are also available that allow desired
features like high efficiency, high fidelity, proofreading and
generation of high yields of long targets. For e.g. a mixture of Pfu
and Taq allows generation of products as long as 35 kb.

12.  All polymerases require free divalent cations –
usually Mg 2+ for activity. Some require Mn 2+
(Tth for RT action).
 Cofactor in the catalytic addition of
deoxynucleoside monophosphates to the 3’
end of the growing DNA chain

13. • A pair of synthetic primers is required to prime DNA
synthesis. A forward and a reverse primer.
• Primers anneal to the flanking regions by complementary-
base pairing (G=C and A=T) using hydrogen bonding.
• The most crucial factor in PCR is the design of the
oligonucleotide primers. Careful design of primers is
required to,
 Obtain desired products in high yields.
 Suppress amplification of unwanted sequences.
 Facilitate subsequent manipulation of the amplified
product.

14.  Base composition:
G+C content between 40% to 60%.
 Length:
18-25 nucleotides long. Members of a primer pair
should not differ in length by >3 bp.
 Complementarity:
The 3’ terminal sequence of one primer should not
be complementary to any site on the other primer.
 Melting temperature (Tm):
The calculated Tm values of a primer pair should not
differ by >5 C.
 3’ termini:
If possible, the 3’ base of each primer should be G or
C.

15.  Wallace rule:
This equation can be used to calculate the Tm of
duplexes 15-20 nucleotides in length in solvents of
high ionic strength (e.g. 1M NaCl).
Tm (in C) = 2 (A+T) +4 (G+C)
 Bolton and McCarthy (1962):
The equation predicts the melting temperature of
oligonucleotides 14-70 nucleotides in length in cation
concentrations of 0.4 M or less:
Tm (in C) = 81.5 C + 16.6 (log10 [K+]) +
0.41 (% (G+C) – 675/n)

16. Many computer programs are available that generate
potentially specific primers whose melting temperatures
have been calculated.
• GeneFisher Interactive Primer Design Tool
• OligoAnalyzer
• Oligocalc
• PCR Optimization Program Helper
• Webprimer

17. The PCR usually consists of a series of 30 to 35 cycles. Most
commonly, PCR is carried out in three steps, often preceded by one
temperature hold at the start and followed by one hold at the end. A
typical PCR cycle has following steps
Denaturation (94-95 C, for ~ 30 s)
The template is denatured by heat
Annealing (55-60 C, for ~ 30 s)
Annealing of oligonucleotide primers to single stranded target
sequences
Elongation (72 C)
Extension of annealed primers by a thermostable polymerase

22. Following PCR, the amplification product can be detected using gel
electrophoresis where visualization of a band containing DNA
fragments of a particular size indicates the presence of the target
sequence in the original starter DNA sample.

23.  Prevent contamination in PCR with exogenous
DNA sequences.
 Always run negative controls.
 Use sterilized filter tips and positive
displacement pipettes.
 Can use UV cabinets.
Although the PCR concept is simple, successful
performance of a PCR reaction depends on a number
of factors

24. For a standard Taq PCR reaction of 30 cycles , the reaction volume
of 25- 50 μl contains
1 pg – 1 μg of DNA
0.5 – 2.5 U of Taq polymerase
0.1 –1μM of each primer
1.5 mM of MgCl2
200 – 250 μM of each dNTP
50 Mm KCl
PCR buffer (Tris-Cl pH 8.3 – 8.8)
When setting up a reaction for the first time with new
template, new primers, new enzyme etc. amplification will be less
than optimal.
Optimization of the reaction to suppress non specific amplification
or enhance the yield of product is required.

25. Template:
The primer / template ratio strongly influences the
specificity of the PCR and should be optimized empirically.
Mg 2+ concentration:
0.5 mM – 5 mM. Influences enzyme activity. The
concentration of free Mg 2+ depends on conc. of dNTPs, free
pyrophosphates and EDTA.
Has to be optimized for every assay.
dNTP concentration:
Imbalanced dNTP mixtures (unequal conc. of four) will
reduce Taq fidelity.

26. Annealing temperature of primers
 Primers have a calculated annealing temperature (e.g. 54°C).
 Temperature must be confirmed practically.
 Temperature steps of 2°C above and below (touch down PCR)
 Use gradient cycler.

27. Problem Explanation Suggested
optimization
Bands are sharp but Insufficient Use different conc. of
faint priming or the two primers/ Mg 2+
extension
Bands in negative Contamination of Make fresh reagents
controls DNA and prevent cross
contamination of
tubes
Undesired products Non specific Decrease the
on gel priming annealing time and
increase temp.

28. Possible causes of Remedies to rectification
non detectable
amplification
Defective reagents Compare yields from fresh & old
reagents
Suboptimal annealing Optimize primer conc., recalculate the Tm
conditions
Suboptimal extension Optimize conc. of dNTPs, DNA, MgCl2,
test pH, use fresh DNA polymerase, add
an enhancer etc.
Ineffective Denaturation Increase time & temp. of denaturation
Primer distance too Use polymerases capable of long
long amplification

29. When multiple amplification products are formed:
 Optimize concentrations of MgCl2, dNTPs,
template DNA and polymerase.
 Use touchdown PCR.
 Verify concentration of primers and optimize if
necessary.
 Carry out nested PCR.

30.  Minute amounts of DNA template may be used from as
little as a single cell.
 DNA degraded to fragments only a few hundred base
pairs in length can serve as effective templates for
amplification.
 Large numbers of copies of specific DNA sequences can
be amplified simultaneously with multiplex PCR reactions.
 Contaminant DNA, such as fungal and bacterial
sources, will not amplify because human-specific primers
are used.
 Commercial kits are now available for easy PCR reaction
setup and amplification.

31.  The target DNA template may not amplify due
to the presence of PCR inhibitors in the
extracted DNA
 Amplification may fail due to sequence changes
in the primer binding region of the genomic
DNA template
 Contamination from other human DNA sources
besides the forensic evidence at hand or
previously amplified DNA samples is possible
without careful laboratory technique and
validated protocols

32.  Generation of probes
 Generation of cDNA libraries
 Production of DNA for sequencing
 Analysis of mutations
 Diagnosis of monogenic diseases (single gene
disorders)
 PCR use in Pre-implantation Genetic Diagnosis (PGD).
 PCR in forensic science
 Comparison of gene expression
 Cloning novel members of protein families using
homology PCR
 Detection of bacteria and viruses

33.  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.
 It enables the scientist to quickly replicate DNA and RNA
on the benchtop.
 PCR and its related applications are rapid and convenient
alternatives to traditional methods such as southern /
northern blotting and molecular cloning.

34. THANK YOU

35.  Heating separates the
double stranded DNA
◦ Denaturation
Heat Cool
 Slow cooling anneals
the two strands
◦ Renaturation

36.  Two primers are supplied in molar excess
 They bind to the complementary region
 As the DNA cools, they wedge between
two template strands
 Optimal temperature varies based on
primer length etc.
Typical temperature from 40 to 60 C

37.  DNA polymerase duplicats DNA
 Optimal temperature 72C