Polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it to a large enough amount to study in detail. PCR was invented in 1984 by the American biochemist Kary Mullis at Cetus Corporation. It is fundamental to much of genetic testing including analysis of ancient samples of DNA and identification of infectious agents. Using PCR, copies of very small amounts of DNA sequences are exponentially amplified in a series of cycles of temperature changes. PCR is now a common and often indispensable technique used in medical laboratory and clinical laboratory research for a broad variety of applications including biomedical research and criminal forensics.

1. Polymerase
chain reaction

2. Identification and detection of a specific
sequence in a genome
LOT of other
sequences in a
genome that we’re not
interested in detecting
SPECIFICITY
The amount of DNA
in samples we’re
interested in is VERY
small
AMPLIFICATION

3. The human
genome is 3.4B
bp
If the bases were
written in
standard type
tape
The tape would
stretch for 5,366
Miles
Identifying a
500bp sequence
in a genome
To be visible on an agarose
gel, need around 10 ng
DNA for fluorescent stain
For a 500-bp product band,
weighing 660 g/mol.bp,
therefore need 10e-9 /
(500*660) = 3.03e-14
moles.Avogadro’s number
= 6.02e23.
Therefore need 1.8e10
copies
To “see” a single “gene”,
the DNA in a sample of
100 cells would have to be
multiplied 180 million
times

4. Real life scenario
In a crime scene, a sample of DNA was found,
however amount of DNA was not enough to be
analyzed

5. PCR=PhotocopierOne billion copies can be synthesized in a matter of hours

6. What it is?
PCR is a
laboratory version
of DNA
Replication in
cells
PCR is a means to
amplify a
particular piece of
DNA Amplify=
making numerous
copies of a
segment of DNA
Kary B. Mullis developed the polymerase
chain reaction (PCR) in 1983

7. How the amplification will be
done?
How you will determine your
target sequence?
How the amplification will be
specific for certain segment?

8. Amplification of a specific target sequence
• PCR copies only a very specific sequence of
genetic code from a template DNA, targeted by
PCR primers
• Knowledge of some DNA sequence
information which flanks the fragment of DNA
to be amplified (target DNA)
• Two synthetic oligonucleotide primers,
complementary to a stretch of DNA to the 3’
side of the target DNA
• DNA polymerase can add a nucleotide only
onto a preexisting 3'-OH group

9. Components of
PCR

10. Overview of PCR Cycle
Denaturation: (95⁰C)
The double-stranded template DNA is
denatured by heating, typically to 95°C, to
separate the double stranded DNA
Annealing: (50-65⁰C)
The reaction is rapidly cooled to an annealing
temperature to allow the oligonucleotide
primers to hybridize to the template
Extension: (72⁰C)
The reaction is heated to a temperature,
typically 72°C for efficient DNA synthesis by
the thermostable DNA polymerase

11. Magnitude of
amplification
“2n”, where n is the number of cycles
The number of DNA copies produced by a
PCR

12. Validate a PCR
• Gel electrophoresis.
• DNA staining dyes (like EtBr) are
applied to the gel to help visualize the
DNA bands using UV transilluminator.
• The presence of a correct size DNA
band indicates that the target sequence was
present and that the PCR has amplified a
correct product.
• Absence of any DNA band indicates that
the target DNA was absent, while the
presence of incorrect size DNA band
indicates production of a spurious product.

13. Variants of
PCR
Conventional PCR
Multiplex-PCR
Nested-PCR
Real-time PCR/quantitative PCR (qPCR)
Touchdown PCR (step-down PCR)
Inverse PCR

14. What’s PCR used for?
Forensic Science Environmental
microbiology
Detection and
quantification of viral
infection
Genetic research Detection of ancient
DNA
Food and agriculture

15. Knowing the steps is not enough…
WHY 2 PRIMERS ARE
USED?
WHY ONLY A
PARTICULAR
TEMPERATURE
USED?
HOW TO DESIGN THE
PRIMERS?
HOW TO GET BETTER
RESULTS?
HOW TO INTERPRET
THE RESULT?
HOW TO REPRESENT
THE RESULT?

16. Biotecnika
initiative to help
you
Module on PCR and
Hybridization
Techniques

17. Course Module
• Basics of PCR -
Introduction,
Principle, Steps
Day 1
• Types of PCR and their
specific applications
Day 2
• Required components of
PCR and chemistry (PCR
template, primer design)
Day 3
• Sample preparation, quality
control and optimization of
PCR parameters
Day 4
• PCR Laboratory
Practices-
Protocols and
Precaution
Day 5

18. Course Module
• Detection of PCR
products and analysis
of results
Day 6
• PCR Technique
applications: PCR-
based genome analysis
Day 7
• Gene expression studies and
Micro RNA assays
Day 8
• Real-time PCR - Fundamentals,
Assay design, Normalization
Day 9
• PCR
Multiplexing
Day 10

19. Course Module
• Quantitative PCR and
bioinformatics
Day 11
• PCR in diagnostic
testing
Day 12
• PCR in COVID 19 Detection
Day 13
• Anomalies of PCR as a research
Tool-Case studies
Day 14

20. Why should I enroll
for this course
• Showcase your competence
• Job advancement
• The course is well designed meets the industry
standard
• Its online- anywhere anytime
• Timings are a plus
• A hard copy of Certificates will be provided on
course completion that adds weightage