4. PCR

Polymerase Chain
Reaction
Nivetha N, PALB 4141

Agril.Microbiology
DEFINITION…
Amplification means making multiple identical copies

(replicates) of a DNA sequence.
DNA amplification
In vivo In vitro
(Cloning) (PCR)
What is PCR ???
The polymerase chain reaction (PCR) is a scientific technique in molecular
biology to amplify a single or few copies of a piece of DNA across several
orders of magnitude, generating thousands to millions of copies of a
particular DNA sequence.
 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.
 The “Reaction” components
It can be thought of as a molecular photocopier.

History…
1953 – Watson and Crick – DNA double helix model
1957 – Arthur Kornberg – 1st DNA Polymerase enzyme
1960s – H. Gobind Khorana - Genetic code and In-vitro DNA synthesis
1969 – Thomas D. Brock – Isolation of Thermus aquaticus
1970 – Klenow – Modified version of DNA Polymerase 1 from E.coli
1971 – Researchers in Khorana’s project - Looking for Repair synthesis

- Kjell Kleppe – Process similar to PCR
1976 – Isolation of Taq Polymerase
1977 – Frederick Sanger – Method to determine sequence of DNA

1983 - Dr. Kary Banks Mullis from Cetus Corporation, Emeryville,
California – Developed PCR
"Beginning with a single

molecule of the genetic material
DNA, the PCR can generate 100
billion similar molecules in an
afternoon. The reaction is easy
to execute. It requires no more
than a test tube, a few simple
reagents, and a source of heat."
In 1993, Mullis was awarded the Nobel

prize in Chemistry along with Michael
Smith for his work on PCR.
Principle…
In-vitro DNA replication
Thermal cycling - cycles of repeated heating and cooling of the

reaction for DNA melting and enzymatic replication of the DNA.
Primers (short DNA fragments) containing sequences complementary

to the target region along with a DNA polymerase, are key
components to enable selective and repeated amplification.
As PCR progresses, the DNA generated is itself used as a template for

replication, setting in motion a chain reaction in which the DNA
template is exponentially amplified.
PCR utilizes the following…
1. Target sequence
2. Two nucleotide primers
3. The four deoxynuclotide triphosphates
4. A heat stable DNA polymerases
 Taq from Thermus acquaticus
 Pfu from Pyrococcus fusiosus
 Vent from Thermus litoralis

5. Mg2+ ions – co factor for DNA polymerase III
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.
Target sequence
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.
• Length of the primer (~ 18 – 22 bp )

 Important in ensuring the amplification of only target sequence.
 Affect Efficiency of PCR ( length indirectly proportional to
efficiency)
• Primers should not have self complementary regions ( to avoid

hairpin formation)
Primer Design
 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.
Nucleotides (dNTPs or deoxynucleotide triphosphates) -
single units of the bases A, T, G, and C, which are essentially "building
blocks" for new DNA strands.
DNA polymerase - a type of enzyme that synthesizes new
strands of DNA complementary to the target sequence. It
moves along the segment of DNA, reading its code and
assembling a copy.
 Taq DNA polymerase (from Thermis aquaticus) – work

at 95ᵒC
 Pfu DNA polymerase (from Pyrococcus furiosus) - higher
fidelity
1) They can generate new strands of DNA using a DNA

template and primers, and
2) They are heat resistant
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
PCR is repeated cycling of three steps:
1. Denature DNA ( 90-98ᵒ C , 2 min)
The DNA is heated to 94° C. This breaks the weak hydrogen bonds that
hold DNA strands together in a helix, allowing the strands to separate
creating single stranded DNA.
2. Primer Annealing ( 40 - 60ᵒ C, 20-40 sec)
The mixture is cooled to 50° C. This allows the primers to bind (anneal) to
their complementary sequence in the template DNA.
3. Extension ( 72ᵒ C, 2 min)
The reaction is then heated to 72° C, the optimal temperature for DNA
polymerase to act. DNA polymerase extends the primers, adding nucleotides
onto the primer in a sequential manner, using the target DNA as a template.
4. Go to Step 1  ( 20 - 35 X )
--------------------------------------------------------------------------------------
- These steps are repeated 20-35 times.
- In PCR, amplification is exponential because for each cycle, the DNA made
in the previous cycles can also serve as template .
Annealing Temperature
• DNA–DNA hybridization – Temperature dependent
phenomenon.
To high temp – no pairing
Low temp – wrong pairing
• Ideal annealing temperature – low enough to allow
hybridization and high enough to avoid amplification
of non target sequences.
• Usually 1-2ᵒC lower than melting temperature.
Melting Temperature
• Dissociation temperature.
• Tm = (4(G+C) + 2(A+T))
• A, T, G, C – No. of Nucleotides in primers.
An automated thermocycler
Variations of PCR
 Modified to suit specific situations and applications
1. Inverse PCR
2. Anchored PCR
3. RT-PCR
4. Site directed mutagenesis using PCR
5. Overlap extension PCR
6. Asymmetric PCR
7. Thermal cycle sequencing PCR
8. Nested PCR
9. Touchdown PCR
10.Hotstart PCR
11.AMRS PCR
Inverse PCR
- Used to amplify sequence located on either side of a segment the
border sequence of which are known.
Anchored PCR
- Used when sequence of only one end of the desired segment or gene is
known.
RT-PCR - Used to amplify RNA sequences into DNA duplexes.
Site Directed Mutagenesis using PCR
Overlap extension PCR
Nested PCR
Asymmetric PCR
- generates single strand copies of one strand of template DNA and
uses one primer at a very low concentration as compared to that of other.
Thermal cycle sequencing PCR

- one primer is used in combination with dideoxynuclotides, the
primer is either radio- or fluorescent labelled. The product of the PCR is
subjected to PAGE for determining the base sequence of the template
DNA.
Touchdown PCR
- to increase the specificity of PCR.
- initiated with high annealing temperature – allow only perfectly
matched primer- template DNA hybrid formation and amplification
- stepwise dropping of annealing temperature – to increase efficiency.
Hotstart PCR
- to prevent non specific amplification.
- Addition of critical component during denaturation.
ARMS PCR
- Amplification refractive mutation system.
Applications of PCR…
To detect mutational in the genes of different individuals,
cells or gametes - data used in disease diagnosis,
population genetics studies etc.,
To construct RAPD maps – ex in Maize, Soybean, Mouse and

Man .
To detect the presence of transgene in transgenic

organisms.
To estimate the frequency of recombination.
To determine the sex of embryo’s.
Generation of single strands for sequencing.

To produce cDNA copies.
Used in fingerprinting.
To analyze the preserved tissues of extinct species –

taxonomy, phylogenetic tree mapping.
Genome mapping.
Prenatal diagnosis of genetic diseases – ex. Sickle cell

anaemia.
Useful in monitoring retroviral infections.
Detection of pathogens which are very difficult to

culture in-vitro.
Advantages of PCR…
1. Highly sensitive technique.

2. The quantity of DNA required for PCR is, low.
3. PCR procedure is very rapid.
4. PCR procedure is relatively very simple, and does not
require specific skills.
5. The purity of the DNA preparation is not critical in case of
PCR.
6. Even degraded DNA can be used as template.
7. Less cost.
8. PCR does not use radioactive chemicals.
9. Allele-specific primers can be designed to directly detect

the presence of specific alleles in individuals; this can be
done with astonishing ease and speed as compared to the
gene cloning procedure.
10. PCR is a highly versatile technique.
11. The PCR technique has been modified in a variety of

ways to achieve specific objectives and to resolve various
problems encountered during PCR.
Limitations of PCR…
 Sequence Information
Amplicon size
 Error rate during amplification
Sensitivity to inhibitors
Contamination
Artefacts
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.
 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.

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Polymerase Chain

Nivetha N, PALB 4141

Amplification means making multiple identical copies

It can be thought of as a molecular photocopier.

1953 – Watson and Crick – DNA double helix model

1957 – Arthur Kornberg – 1st DNA Polymerase enzyme

1960s – H. Gobind Khorana - Genetic code and In-vitro DNA synthesis

1969 – Thomas D. Brock – Isolation of Thermus aquaticus

1970 – Klenow – Modified version of DNA Polymerase 1 from E.coli

1971 – Researchers in Khorana’s project - Looking for Repair synthesis

1976 – Isolation of Taq Polymerase

1977 – Frederick Sanger – Method to determine sequence of DNA

"Beginning with a single

In 1993, Mullis was awarded the Nobel

In-vitro DNA replication

Thermal cycling - cycles of repeated heating and cooling of the

Primers (short DNA fragments) containing sequences complementary

As PCR progresses, the DNA generated is itself used as a template for

2. Two nucleotide primers

3. The four deoxynuclotide triphosphates

4. A heat stable DNA polymerases

 Taq from Thermus acquaticus

 Pfu from Pyrococcus fusiosus

 Vent from Thermus litoralis

• Length of the primer (~ 18 – 22 bp )

• Primers should not have self complementary regions ( to avoid

 Taq DNA polymerase (from Thermis aquaticus) – work

1) They can generate new strands of DNA using a DNA

Thermal cycle sequencing PCR

To construct RAPD maps – ex in Maize, Soybean, Mouse and

To detect the presence of transgene in transgenic

To estimate the frequency of recombination.

To determine the sex of embryo’s.

Generation of single strands for sequencing.

To analyze the preserved tissues of extinct species –

Prenatal diagnosis of genetic diseases – ex. Sickle cell

Useful in monitoring retroviral infections.

Detection of pathogens which are very difficult to

1. Highly sensitive technique.

9. Allele-specific primers can be designed to directly detect

10. PCR is a highly versatile technique.

11. The PCR technique has been modified in a variety of

 The speed and ease of use, sensitivity, specificity and robustness of

 It enables the scientist to quickly replicate DNA and RNA on the

 PCR and its related applications are rapid and convenient

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