The document describes polymerase chain reaction (PCR), a technique used to amplify a single copy 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. PCR requires a DNA template, DNA polymerase enzyme, primers, nucleotides, and magnesium ions. It works by cycling between heating and cooling steps to denature the DNA, anneal primers, and extend the DNA. PCR can amplify very small amounts of DNA and is widely used in applications like pathogen detection, DNA fingerprinting, and molecular archaeology. Real-time PCR monitors fluorescence during amplification to quantify DNA at each cycle.

1. POLYMERASE CHAIN REACTION
Noida Institute of Engineering and Technology
(Pharmacy Institute)
Greater Noida
Presented by :-
ANMOL KANDA
M.Pharm(Pharmacology)
Submitted to :-
Dr. Saumya Das
Associate Professor
NIET (Pharmacy Institute)
Greater Noida

2. TITLE -
PCR
(POLYMER
ASE CHAIN
REACTION)

3. INDEX
1.
INTRODUCTION
2.
COMPONENTS
3.
PROCESS/PROC
EDURE
4.
ADVANTAGES
&
APPLICATIONS
5.
UPGRADED
FORM OF PCR

4. INTRODUCTION
WHAT IS PCR?
It was first developed in the year 1983 by KARY
MULLIS.
In the Year 1993 KARY MULLIS Was awarded
with Nobel Prize in Chemistry for his work on
PCR.
Polymerase chain reaction is the branch of
molecular biology , which amplify a single or
few copies of a segment of DNA across several
orders of Magnitude, generating thousands to
millions copies of a particular DNA sequence.

5. General components of PCR
1. DNA TEMPLATE-
2. DNA POLYMERASE ENYZME
3. PRIMERS
4. NUCLEOTIDES
5. MAGNESIUM ION
https://www.bag-diagnostics.com/en/technology_pcr.html
1. DNA TEMPLATE-
• DNA template is a target sequence.
• DNA template is a DNA Molecule that contains DNA region ( segment ) to be amplified
https://slidemodel.com/templates/dna-strands-powerpoint-template/

6. • 2. PRIMERS-
• Primers are synthetic DNA strand of about 18 to 25 Nucleotide complimentary
to 3’ end of the template strand.
• DNA Polymerase starts synthesizing new DNA from the 3’ end of the primer.
• Two primers must be designed for PCR.
1. Forward Primer
Complimentary to the 3’ end of anti-sense strand.
2. Reverse Primer
Complimentary to the 3’ end of sense strand.
https://www.addgene.org/protocols/primer-design/

7. • 3. DNA POLYMERASE-
• DNA Polymerase sequentially adds Nucleotides complimentary to template
strand at 3’-OH of the bound primers and synthesizes new strands of DNA.
• The most commonly used DNA Polymerase enzyme Example
1. Taq DNA Polymerase enzyme
Thermus aquaticus , a thermophilic bacteria .
High temperature stability and work optimally at temp. 72 degree Celsius .
2. Pfu DNA polymerase enzyme
Pyrococcus furiosus .
Show higher fidelity to accurately adding complimentary nucleotide .

8. • 4. NUCLEOTIDES ( dNTPs )
• Nucleotides are “building blocks” for new DNA strands and essential
for reaction.
• EXAMPLE- Adenine (A), Guanine(G), Cytosine (C) , Thymine (T).
• 5. MAGNESIUM ION.
• Mg2+ ions act as a co-factor for DNA Polymerase enzyme and hence
required for reaction.
• It also affects primer annealing and template denaturation.
• Excess of magnesium gives non-specific amplification while low
magnesium yields lesser amount of desired product.

9. STEPS IN INVOLVED IN PCR
There are three major steps in a PCR, which are repeated for 30 or 40
cycles. This is done on an automated cycler ,which can heat & cool the
tubes with the reaction mixtures in a very short time.
STEP 1- Denaturation at 94 Celsius
The reaction mixture is heated at 94 Celsius .
This denaturation cause separation of DNA double strand , now each
strand act as a template for synthesis of complimentary strand .
This step work for about 15 – 25 seconds .
https://www.goldbio.com/goldbios-pcr-overview

10. • STEP 2- Annealing at 54 Celsius
• Cooling of reaction mixtures after denaturation step at 54 Celsius.
• This cause hybridization ( annealing ) of primer to separated strand of DNA (
Template)
• Guanine cytosine content should be sufficient for stable binding with
template.
• This step work for 15 seconds.
http://www.cryst.bbk.ac.uk/pps97/assignments/projects/borek/Domina/page3a.html

11. • STEP 3- Extension at 72 Celsius
• The reaction mixture is heated to 72 Celsius which is ideal temperature for the Taq-
polymerase enzyme.
• Polymerase adds nucleotides ( dNTPs) complimentary to template on 3’-OH of primer
there by extending the new strand.
https://www.goldbio.com/goldbios-pcr-overview
• STEP 4- Final hold
• First three steps are repeated 30-40 times to produce millions of exact copies of the
target DNA.
• Once several cycles are completed , during the hold step , 4 to 15 Celsius temperature is
maintained for short-term storage of the amplified DNA template.

12. ADVANTAGES & APPLICATION OF PCR
• ADVANTAGES-
a) Small amount of DNA required per test.
b) Result obtained more quickly.
c) Not necessary to use radio-active material for PCR.
d) PCR can be used to detect point mutation.
• APPLICATION –
a) Detection of pathogens
b) Prenatal diagnosis
c) DNA finger-printing.
d) Drug discovery
e) Molecular archaeology
https://www.clinisciences.com/es/comprar/cat-conventional-pcr-3473.html

13. REAL – TIME PCR
REAL – TIME PCR MONITORS THE FLUORESCENSE EMITTED DURING THE REACTION AS
AN INDICATOR OF AMPLIFICATION PRODUCT AT EACH CYCLE (in real time) AS OPPOSED
TO THE END POINT DETECTION.
https://www.bio-rad.com/en-in/applications-technologies/introduction-qpcr-instrumentation?ID=LUSO5YMNI
THIS KINETIC PCR ALLOWS FOR DATA COLLECTION AFTER EACH CYCLE OF PCR INSTREAD
OF ONLY AT THE END OF THE 20 TO 40 CYCLES.

14. • REFERNCE
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4102308/
• PCR STRATEGIES 1st Edition - June 27, 1995,Editors: Michael Innis, David Gelfand, John Sninsky , eBook ISBN:
9780080538549
• https://www.genome.gov/about-genomics/fact-sheets/Polymerase-Chain-Reaction-Fact-Sheet

15. THANK YOU