The document provides an overview of the Polymerase Chain Reaction (PCR) technique, detailing its basic concept, major steps (denaturation, annealing, and extension), and its applications in various fields such as gene expression analysis and disease diagnosis. It also introduces quantitative real-time PCR, emphasizing its advantages, such as real-time monitoring and accurate quantification of DNA amplification. Additionally, the document discusses important aspects of primer design and software tools available for designing primers.

1. .
PRESENTED BY
NAVEED UL
MUSHTAQ
1
POLYMERASE CHAIN
REACTION
NAVEED UL MUSHTAQ
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2. CONTENTS:
 Basic concept of PCR.
 Steps in PCR.
 Quantitative real time polymerase chain
reaction.
 Fluorescent dyes and probes.
 Advantages real-time PCR.
 Real-time PCR primer
 Primer design software
2

3. INTRODUCTION
• The polymerase chain reaction (PCR) is a scientific
technique in molecular biology to amplify a single or a
few copies of a piece of DNA across several orders of
magnitude, generating thousands to millions of copies of
a particular DNA sequence.
• There are three major steps involved in the PCR
technique: denaturation, annealing, and extension.

4. INTRODUCTION
• Polymerase Chain Reaction was developed in 1984 by
the American biochemist, Kary Mullis. Mullis received
the Nobel Prize and the Japan Prize for developing PCR
in 1993.
• However the basic principle of replicating a piece of
DNA using two primers had already been described by
Gobind Khorana in 1971.
• The polymerase chain reaction is a powerful technique
that has rapidly become one of the most widely used
techniques in molecular biology because it is quick,
inexpensive and simple.

5. Basic concept of PCR
• As the name implies, it is a chain 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 polymerases, 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), thymine (T), cytosine (C) and guanine
(G).

6. Thermal pool in Yellowstone
National Park (Thermus
aquaticus)

7. 7
NEW PCR OLD
PCR

8. Real time pcr

9. Steps in PCR
• There are three major steps involved in the PCR
technique:
• Denaturation,
• Annealing, and
• Extension.

10. Steps in PCR
• In step one; the DNA is denatured at high temperatures (from
90 - 97 degrees Celsius).
• In step two, primers anneal to the DNA template strands. The
annealing phase happens at a lower temperature, 50-60°
• In step three, extension occurs at the end of the annealed
primers to create a complementary copy strand of DNA. The
extension of the primers by Taq polymerase occurs at approx
72°C for 2-5 minutes.
• DNA polymerase I cannot be used to elongate the primers as
one would expect because it is not stable at the high
temperatures required for PCR.

11. Quantitative real time
polymerase chain reaction
• Quantitative real time polymerase chain reaction is a
technique based on the PCR, which is used to amplify
and simultaneously quantify a targeted DNA molecule.
• Traditionally, PCR is performed in a tube and when the
reaction is complete the products of the reaction (the
amplified DNA fragments) are analyzed and visualized
by gel electrophoresis.
• However, Real-Time PCR permits the analysis of the
products while the reaction is actually in progress.

12. Quantitative real time
polymerase chain reaction
• This also facilitates the quantitation of the DNA i.e. used
to measure the quantity of a PCR product.
• This is achieved by using various fluorescent dyes which
react with the amplified product and can be measured by
an instrument.
• Fluorescent signal in direct proportion to the number of
PCR product molecules (amplicons) generated.
• Data collected in the exponential phase of the reaction
yield quantitative information on the starting quantity of
the amplification target.

13. Plateau at cycle 25 due to inhibitory effect
of accumulation of pcr product on
polymerase

14. Ct is the number of cycles
required for the fluorescent signal
to cross the threshold.

15. Fluorescent dyes and
probes
• QRT-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.

19. Advantages real-time PCR
• Ability to monitor the progress of the PCR reaction as it
occurs in real time.
• Ability to precisely measure the amount of amplicon at
each cycle, which allows highly accurate quantification of
the amount of starting material in samples.
• Amplification and detection occurs in a single tube,
eliminating post-PCR manipulations.
• Gene expression analysis.
• Cancer research.
• Disease diagnosis and management.
• Food testing.
• Animal and plant breeding.

20. Real-time PCR primer
• Good primer design is one of the most important
parameters in real-time PCR.
• In general, primers should be 18–24 nucleotides in length
• Primer pairs should have compatible melting
temperatures (within 5°C) and contain approximately
50% GC content

21. Primer design software
• Primer design software programs Software can
automatically evaluate a target sequence and design
primers for it based on the criteria
• OligoPerfect designer
• Primer Express software
• Vector NTI

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