All about polymerase chain reaction. detailed description and explanation of instrumention, procedure, advantages, disadvantages. Also types of RtPcr.. graphical representation. explained with appropriate figrues.

1. Bhupal Noble’s University
M.Pharm(Pharmacology)
1ST Semester (2022 - 2023)
Polymerase Chain Reaction
Name: Joshi Poonam
Roll No: 3
Subject Teacher: Dr. J.S.Vaghela Sir

2. Content
 What is PCR?
 Instrument
 Requirements
 Principle
 RT-PCR
 RealTime PCR
 Advantages
 Disadvantages
 Applications

3. Polymerase Chain Reaction
 PCR technique is invented by Kary Mullis in
1984.
 Polymerase Chain Reaction (PCR) is technique
for generating large quantities of a specified
DNA
 PCR is a cell free amplification technique for
synthesizing multiple identical copies of any
DNA of interest by in vitro method.
 PCR is “photocopier”

4. Instrument
 The appratus is called
Thermal Cycler or DNA
Amplifier
 The device has thermal
block with holes where
tubes holding reaction
mixture are inserted.
 Cycler then raises and
lower the temperature
of the blocks in pre
programmed steps

5. Requirements
 DNA Template (100-35,000) bp in length
 Primers (synthetic oligonucleotides of 17-30 nucleotide
length)
 Taq polymerase that can withstand at temperature
upto 95ºC
 Deoxynucleoside triphosphate (dNTPs) {dATP, dCTP,
dGTP, dTTp}
 Buffer Solution MgCl2,Tris-HCl, KCl, Gelatin
 PCR Master Mix

6. Principle
PCR is based on the three steps:
 Denaturation
 Renaturation or Annealing
 Synthesis

7. Denaturation
 The reaction mixture is heated upto 96ºC for1minute which
cause strands of template DNA to unwound and separate by
breaking of Hydrogen bond between two strands resulting in
single stranded DNA molecule.

8. Annealing
 Reaction mixture is then cooled to 54ºC, which
cause annealing of primer to separated strand
of DNA

9. Synthesis
 Temperature of reaction mixture is then raised to 68-
72ºC.
 Taq polymerase enzyme which is thermostable at higher
temperature bind to the primer and synthesis a new
DNA.

10. Graphical Representation

11. Reverse Transcription PCR
 PCR can be used to amplify sequences corresponding
to RNA.
 This amplification is accomplished by first converting
RNA into complementary DNA(cDNA) with the use of
enzyme Reverse Transcriptase
 This technique is commonly used in molecular biology
to detect RNA expression.
 It was introduced in 1977
 In recent COVID times this technique was widely used
for detection of corona virus.

12. RT-PCR Principle
One Step RT-PCR
• In this all reaction component are mixed in one tube in
common buffer prior to initiation of reaction.
• The resulting cDNA cannot be used for detecting
multiple messages from single RNA sample as in two
step RT-PCR
• It offers minimal sample handling, reduced bench time,
and closed tube reaction thus minimize the possibility of
contamination.
• One step RT-PCR offers simplicity and convenience.

13. RT-PCR Principle contd..
 Two Step RT-PCR
 RNA is first transcripted to cDNA using Reverse
Transcriptase Enzyme
 The resulting cDNA is used as template for subsequent
PCR amplification, using primer specification for one or
more gene.
 As only a portion of cDNA is used remaining cDNA
can be stored for future use.

14.  Allows freedom in selecting RT enzyme and qPCR
reagents separately.
 Useful for controlling sequence representation
efficiency and optimization of difficult RT- qPCR
reactions.
 Drawback is that it require an extra open tube
step, more pipetting manupulations,and longer
hands on time.
 This lead to greater variabilty and risk of
contamination.

15. Schematic Representation of one
step PCR & two step PCR

16. Real Time PCR
 Modification of conventional PCR where
product accumulation is measured/
visualized in “real time” as the reaction
progress after each cycle.
 Real Time PCR is a simple method to
determine the amount of target sequence
or gene that is present in sample.

17. Advantages
 Specific and Sensitive
 Fast and safe process (can be done <1day)
 Radioactive material not required
 Small amount of DNA is required per test
 Detection of bacteria and viruses

18. Disadvantages
 Setting up and running requires high
technical skills
 High equipment cost
 DNA contamination
 Taq polymerase is expensive
 Internal Control
 False reaction

19. Applications
 Diagnosis of cancer, genetic abnormalities.
 Detect newly emerging diseases. Ex covid, and its
variants
 Detection of Phytopathogens
 Clinical Quantification and Genotyping
 Generating cDNA libraries
 DNA fingerprinting
 Genomic Cloning
 Gene expression studies
 Drug discovery
 Pre-natal diagnosis