Document describes detailed about polymerase chain reaction including its procedure, principle, advantages, disadvantages, and its application

1. Polymerase chain reaction.
Presented by- Sk Aziz uddin
Submitted to- Prof. Sandhya Bawa
Course- M.Pharm
Department- Pharmaceutical Analysis.
Batch- 2020-2022
College Name- Jamia Hamdard.

2. Index
 What is PCR( Definition, principle)
 History of PCR
 Thermal cycler
 Components of PCR
 Three basic steps
 Application of PCR
 Advantages of PCR
 Disadvantages of PCR

3. PCR( Polymerase chain reaction)
 Definition- The Polymerase chain
reaction(PCR) is a laboratory( in vitro)
technique for generating large quantities
of a specified DNA. Obviously PCR is a
cell- free amplification technique for
synthesizing multiple identical
copies(billions) of any DNA of interest.
 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.

4. Principle of PCR
 The double- stranded DNA of interest
is denatured to separate into two
individual strands, each strand is then
allowed to hybridize with a primer(
renaturation). The primer-template
duplex is used for DNA synthesis( the
enzyme DNA polymerase). These three
steps- denaturation, renaturation, and
synthesis are repeated again and again
to generate multiple forms of target
DNA.

5. Exponential Amplification
 The DNA sequence between primers
doubles after each cycle.

6. History of PCR
 The polymerase chain reaction(PCR) was
originally developed in 1983 by the American
biochemist Kary Mullis.
 He was awarded the Nobel prize in chemistry
in 1993 for his pioneering work.
 In 1985 Cetus Corp, scientist isolate Thermo
stable Taq polymerase ( from aquaticus),
which revolutionized PCR.

7. Thermal cycler-:
 Also called PCR machine or DNA amplifier.
 The thermo cycler is a laboratory apparatus
most used to amplify segments of DNA via
polymerase chain reaction(PCR).
 Provides favourable environment.
 Regulates temperature during cyclic
programs.
 All components are placed in a thin walled
tube and then these tubes are placed in the
PCR thermal cycler.

8. PCR components in Thermal
cycler

9. Working principle of thermo
cycler
 Thermocyclers increases and decreases
the temperature of the block in
discrete, pre-programmed steps.
 Performs denaturation and annealing
and extension of samples in repeated
manner.
 Thermocyclers amplify DNA and RNA
samples by the process of polymerase
chain reaction.

10. Instrumentation
 Thermocyler has a thermal block with
holes where tubes holding the reaction
mixtures can be inserted.
 Equipped with a heated lid that presses
against the lids of the reaction tubes.
 Lid prevents condensation of water
from the reaction mixtures on the
insides of the lids.

11. Instrumentation of thermocycler
 Wells- 4-384(96 mostly)
 Temperature range- 0-1000 c
 Heating rate- 3-70 c/sec
 Cooling rate- 3-70 c/sec
 Volume-10-100µl

12. Instrumentation contd.....

13. Five important features of standard
thermal cycler.
 Sample throughput-From different type of
thermocycler models, choose the sample
through put as per your lab need. For small
number of samples, so called personal PCR
machines that sit unobtrusively on your
bench top or desk may be perfect. They have
higher speed, because their diminutive sizes
facilitates faster changes in temp. On the
other hand high- throughput PCR holds as
many sample as possible. It can be used in
thermocycler models that can be networked
together and controlled from one instrument.

14. Important features contd...
 Heating- block format- The heating block
in which the PCR sample tubes incubate is
available in different formats. The first
considerations are how many samples the
block holds and sample volumes. The
most basic thermo cyclers may offer only
one choice.
 Thermal gradient- Programmable thermal
gradients are useful for finding optimal
PCR conditions( e.g. In primer annealing).
Simple optimization usually requires only
the basic gradient function of a simple,
single gradient across the heating block.

15. Important features contd...
 Maximum ramp rate.- A thermo cycler's
maximum ramp rate is the maximum rate
at which its heating block can change
temperature. A faster ramp rate is a good
thing. Usually the faster ramp rates are on
the order of 50 c or 60 c per second.
Another important consequence of the
ramp rate is the length of your entire
protocol, because ramp rate determines
how long it will take your thermocycler to
complete a specified number of cycles.

16. Important features contd...
 High- tech lids- specialized
thermocycler lids can head off two
problems that may otherwise wreak
havoc with your PCR reactions-
especially if you are using smaller
sample volumes. These problems will
have a greater effect on the
concentration of reaction components
when using small volumes.

17. Components of PCR
 DNA template- That contains the DNA
region ( target) to amplify.
 Taq polymerase- A DNA polymerase
that is heat resistant, so that it can
remain intact during the DNA
denaturation process.
 Two primers- That are complimentary
to the 3’ ends of each of the sense and
anti-sense strand of the DNA target and
needed to initiate DNA synthesis.

18. Components of PCR
 Deoxynucleoside triphosphates-: The building
blocks from which the DNA polymerase
synthesizes a new DNA strand.
 Buffer solution-: Providing a suitable chemical
environment for optimum activity and stability
of the DNA polymerase. The standard PCR
buffer contains; Mgcl2 Tris-HCL, KCL, Gelatin
or Bovine Serum Albumin
 Bivalent cations-: Magnesium or manganese
ions, generally Mg+2 is used, but Mn+2 can be
used for PCR mediated DNA amplification.

19. PCR master mix
 PCR master is a pre mix ready to use
solution containing;
 Taq DNA polymerase
 dNTPs
 MgCL2
 Reaction buffer at optimal
concentrations for efficient
amplification of DNA templates by
PCR

20. Three basic steps
1. Denaturation(960 c)-Heat the reaction
strongly to separate or denature, the
DNA strands. This provides single-
stranded template for the next step.
2. Annealing(55-65o c)- Cool the reaction
so the primers can bind to their
complimentary sequences on the single-
stranded template DNA.
3. Extension(720 c)- Raise the reaction
temperatures so Taq polymerase extends,
the primers synthesizing new strands of
DNA.

21. Steps in PCR-:

22. Application of PCR
 Medical Application:-
 Genetic testing for presence of genetic
disease mutations.
 Detection of disease causing genes in
suspected parents who act as carrier.
 Study of alteration to oncogenes may help
in customization of therapy.
 Can also be used as part of a sensitive test
for tissue typing, vital to organ
transplantation genotyping of embryo.
 Helps to monitor the gene therapy.

23. Application of PCR
 Infectious disease application-:
 Analyzing clinical specimens for the
presence of infectious agents, including
HIV , hepatitis, malaria, tuberculosis
etc.
 Detection of new virulent subtypes of
organism that is responsible for
epidemics.

24. Application of PCR
 Forensic application-:
 Can be used as a tool in genetic
fingerprinting.
 This technology can identify any one
person from millions of others in case
of crime scene, paternity testing etc.

25. Application of PCR
 Forensic application-:

26. Application of PCR
 Research and molecular genetics-:
 Helps to compare the genomes of two
organisms and identify the difference
between them.
 In phylogenetic analysis, minute
quantities of DNA from any source such a
fossilized material, hair, bones,
mummified tissues.
 In human genome project for aim to
complete mapping and understanding of
all genes of human beings.

27. Advantages of PCR
 Automated, fast, reliable( reproducible)
results.
 Contained( less chance of
contamination)
 High output
 Sensitive
 Broad use
 Defined, easy to follow protocols

28. Disadvantages of PCR
 Setting up and running requires high
technical skills.
 Not easy to quatitate results.
 High equipment cost.
 High sterile environment should be
provided.

29. Acronyms
 PCR- polymerase chain reaction
 DNA- deoxy ribonucleic acid
References-: Biotechnology book by
satyanarayan
Google slide share
YouTube- Khan academy

30. THANK YOU