Polymerase chain reaction. 1. Denaturation. 2. Annealing. 3. Extension.

1. DAVANGERE UNIVERSITY
MICROBIOLOGY SEMINAR
ON THE TOPIC
PCR
SUBMITTED BY :
Ponnanna. M.B
M.Sc. Microbiology.

3. CONTENTS
INTRODUCTION
HISTORY
WHAT IS PCR?
WORKING PRINCIPLE OF PCR
COMPONENTS OF PCR
STEPS INVOLVED IN PCR
TYPES OF PCR
APPLICATIONS OF PCR
ADVANTAGES & LIMITATIONS OF PCR
SUMMARY
CONCLUSION
REFERENCES

4. INTRODUCTION
PCR is an abbreviation for ‘polymerase chain reaction’. It is
a technique used to amplify a single copy or few copies of
segments of DNA across several orders of magnitude,
generating thousands of copies of DNA sequences. It is an
easy, cheap and reliable way to repeatedly replicate a
focused segment of DNA. A concept which is applicable to
numerous fields in modern biology and related sciences.
PCR is probably the most widely used technique in
molecular biology, criminal forensics, molecular
archaeology and in clinical and biomedical research.

5. HISTORY
1983: Kary Mullis developed the polymerase chain reaction
[PCR] technique.
The process similar to PCR was first described by Kjell Kleppe
and Nobel laureate Har Gobind Khorana in 1971, which allows
the amplification of specific DNA sequences.
1993: Kary Mullis was awarded the Nobel Prize in Chemistry for
his invention of PCR. Michael Smith also shared the Nobel Prize
with Kary Mullis for his work in developing site-directed
mutagenesis.
1986: Purified Taq polymerase was first used in PCR.
1988: DNA fingerprinting was first used for paternity testing.

6. • What is PCR?
PCR is an exponentially progressing synthesis of the defined
target DNA sequences in vitro.
PCR [Polymerase chain reaction]
• Polymerase- The only enzyme used in this reaction is
DNA polymerase.
• Chain- The products of the first reaction becomes the
substrates of the following one, and so on.
• Reaction components- Target DNA, pair of primers,
thermostable DNA polymerase, Mg++ ions, buffer
solution.

7. Working principle of PCR
As the name implies, it is a chain reaction, a small fragment of
the DNA strand of our interest needs to be identified, which
serves as the template for producing the primers that initiate the
reaction. One DNA molecule is used to produce two copies,
then four, then eight and so forth. This continuous doubling is
accomplished by specific proteins known as polymerase
enzymes, that are able to string together individual DNA
building blocks to form long molecular strands. To do their job
polymerases require a supply of DNA building blocks, i.e., the
nucleotides consisting of the four bases adenine (A), guanine
(G), thymine (T) and cytosine (C). They also need a small
fragment of DNA known as the primer, to which they attach the
building blocks as well as a longer DNA molecule to serve as a
template for constructing the new strand. If these three
ingredients are supplied, the enzymes will construct exact
copies of the templates.

10. Basic Requirements for PCR
• DNA Template: It is made up of double helix of two
complimentary strands. Each strand of the duplex acts as a
template for the synthesis of new double helix.
• Primer: It is a short strand of RNA or DNA that serves as a
starting point for DNA synthesis. It is required for DNA
replication.
• DNA Polymerase : Enzymes that synthesize DNA molecules
from deoxyribonucleotides , the building blocks of DNA. [
Taq polymerase is used in this process]
• dNTPs: deoxynucleotide triphosphates are the monomeric
substrates for the polymerization reaction. It is a mixture of
the four monomeric units, dATP, dTTP, dCTP and dGTP that
will ultimately make up the DNA that is polymerized during
PCR.
• Divalent cations: All thermostable DNA polymerases
require free divalent cations- usually Mg2+ for their activity.

11. • Buffer solution : It is necessary to create optimal
conditions for activity of Taq DNA polymerase.[ Tris- Hcl]
• # Pfu- Pyrococcus furiosus
 # Vent- Thermococcus
litoralis litoralis.

12. DNA Thermal cycler
The thermal cycler (also known as a thermocycler) is a
laboratory apparatus which is used to amplify segments of
DNA via the polymerase chain reaction(PCR). This
machine can be programmed to carry out heating and
cooling of samples over number of cycles.

13. Steps involved in PCR.
 DENATURATION
The reaction mixture is heated to a temperature between 94-
96ºC so that ds DNA is denatured into single stranded by
disrupting the hydrogen bonds between complementary
bases. This results in two single strands of DNA, which acts
as a template for the production of new strands of DNA.
Duration of this step is 30-60seconds.

14.  ANNEALING
During this stage, the reaction temperature is
lowered to 55–65 °C for 20–30 seconds, allowing
annealing of the primers to each of the single-
stranded DNA templates. Primers serve as the
starting point of DNA synthesis. Two separated
strands of DNA are complementary and run in
opposite directions , as a result there are 2 primers- a
forward and reverse primer.

15. Extension/elongation:
The temperature at this step depends on the DNA
polymerase used; the optimum activity temperature for
the thermostable DNA polymerase of Taq (Thermus
aquaticus) polymerase is approximately 75–80°C.
Though a temperature of 72 °C is commonly used with
this enzyme in this step, the DNA polymerase
synthesizes a new DNA strand complementary to the
DNA template strand by adding free dNTPs from the
reaction mixture that are complementary to the template
in the 5'-to-3' direction, condensing the 5'-phosphate
group of the dNTPs with the 3'-hydroxy group at the end
of the elongating DNA strand.

16. These three processes of thermal cycling are repeated several
times to produce many copies of DNA sequences.

18. Quantitative PCR (qPCR / real-time PCR):
Used to measure the quantity of a target sequence
(commonly in real-time). It quantitatively measures starting
amounts of DNA, cDNA, or RNA. Quantitative PCR is
commonly used to determine whether a DNA sequence is
present in a sample and the number of its copies in the
sample. Quantitative PCR has a very high degree of
precision. Quantitative PCR methods use fluorescent dyes,
such as Sybr Green, EvaGreen or fluorophore-containing
DNA probes, such as TaqMan, to measure the amount of
amplified product in real time.

20. Reverse Transcription PCR (RT-PCR):
For amplifying DNA from RNA, reverse transcriptase
reverse transcribes RNA into cDNA, which is then amplified
by PCR. RT-PCR is widely used in expression profiling, to
determine the expression of a gene or to identify the
sequence of an RNA transcript, including transcription start
and termination sites. If the genomic DNA sequence of a
gene is known, RT-PCR can be used to map the location of
exons and introns in the gene.

22. Nested PCR:
It increases the specificity of DNA amplification by
reducing background due to non-specific amplification of
DNA. Two sets of primers are used in two successive PCRs.
In the first reaction, one pair of primers is used to generate
DNA products, which besides the intended target may still
consist of non-specifically amplified DNA fragments. The
product(s) are then used in a second PCR with a set of
primers whose binding sites are completely or partially
different and located 3' of each of the primers used in the
first reaction.
Nested PCR is often more successful in specifically
amplifying long DNA fragments than conventional PCR,
but it requires more detailed knowledge of the target
sequences.

24. Multiplex-PCR:
It consists of multiple primer sets within a single PCR
mixture, it produces amplicons of varying sizes that are
specific to different DNA sequences. By targeting
multiple genes at once, additional information may be
gained from a single test-run that otherwise would
require several times the reagents and more time to
perform.
Annealing temperatures for each of the primer sets
must be optimized to work correctly within a single
reaction, and amplicon sizes. That is, their base pair
length should be different enough to form distinct bands
when visualized by gel electrophoresis.

26. Hot start PCR:
It is a technique that reduces non-specific amplification
during the initial set up stages of the PCR. It may be
performed manually by heating the reaction components
to the denaturation temperature (94°C) before adding
the polymerase. Specialized enzyme systems have been
developed that inhibit the polymerase activity at
ambient temperature, either by the binding of an
antibody or by the presence of covalently bound
inhibitors that dissociate only after a high-temperature
activation step.
When temperature raises for amplification at 72ºC, the
specific antibodies detaches from the DNA polymerase
and amplification begins with great specificity.

28. Inverse PCR:
It is commonly used to identify the flanking
sequences around genomic inserts. It involves a
series of DNA digestions and self ligation, resulting
in known sequences at either end of the unknown
sequence.

31. Advantages of PCR:
Small amount of DNA sample is required.
Amplified copies of DNA are obtained quickly .
Use of radioactive substances is not necessary.
PCR is more precise in determining the size of alleles
essential for some disorders.
Limitations of PCR:
It is a sensitive technique, prone to contamination from
extraneous DNA , leading to false positive result.
Cross contamination between samples is the potential
problem..
Instrument and reagents are expensive, hence cannot be
afforded by small laboratories.

32. SUMMARY
PCR is the technique that amplifies a small amount of
known sequence of DNA into many copies within short
period of time. The process involves three steps
denaturation, annealing and extension along with addition
of several components required for the reaction. There are
various types of PCR working under different principles
and has a wide range of application in the field of
molecular biology.

33. CONCLUSION
PCR is one vital process to amplify specific DNA fragments
from small amounts of DNA sample. The speed and ease of
use, sensitivity, specificity and robustness of PCR has
revolutionized molecular biology and made PCR the most
widely used technique with great spectrum of research and
diagnostic applications.

34. REFERENCES
• Clark, D. P. and Pazderinik, N. J. 2009.
Biotechnology. Elsevier Academic Press, US,750 pp.
• Hughes, S. and Moody, A. 2007. PCR. Schion
publishing limited, UK, 348 pp.
• Modi, H. A. 2009. Microbial Biotechnology. Pointer
publishers, India, 355 pp.
• Schleif, R. 1993. Genetics and molecular biology, 2nd
edn. John Hopkins University press, London, 685 pp.
• Walker, J. M. and Raply, R. 2009. Molecular Biology
and Biotechnology, 5th edn. RSC publishers, UK,
585 pp.
• Watson, J. D., Caudy, A . A., Myers, R. M. and
Witkowski, J. A. 2007. Recombinant DNA, 3rd edn.
Cold spring harbour press, New York, 474 pp.

35. THANK YOU.