The document summarizes polymerase chain reaction (PCR), including its history, principles, types, and applications. It describes how PCR was invented in 1983 by Kary Mullis, allowing for the amplification of specific DNA sequences. The basic steps of PCR involve denaturation of DNA, annealing of primers, and extension of new strands by DNA polymerase. Various types of PCR are discussed, such as real-time PCR, reverse transcriptase PCR, and nested PCR. The document explains that PCR has many applications, including diagnosis of infectious diseases and detection of genetic variations.

1. Polymerase chain reaction
Presented by: Vikash Kumar
M.Sc. Medical Lab Science(2nd year)
Moderated by :Mrs. Bhumika Upadhyay

2. content
• Introduction
• Short History of PCR
• Principle of PCR
• Working of PCR
• Types of PCR
• Application

3. History
• Kary Mullis was an
American biochemist.he
invented this technique in
1983,he was awarded Nobel
prize in 1993.
• His invention became a
central technique
in biochemistry and molecular
-biology,

4. Introduction
• The polymerase chain reaction or PCR is an in
vitro technique.
• It is like xerox machine for gene copying.
• PCR is a method of amplifying specific DNA
sequences & RNA sequences.
• It can be used to very quickly & efficiently
produce millions copies of a single DNA
segment.

5. Working principle of PCR
• One DNA molecule is used to produce
two copies, then four, then eight and so
on.
• This continuous doubling is accomplished
by specific proteins known as polymerase
enzyme that are able to string together
individual DNA building blocks to form
long molecular strands.

6. Reagents Needed
• Target DNA molecule (DNA sample which you want to
amplify)
• A pair of DNA primer
• Forward primer (One primer binds to the 5’ end of one
of the DNA strands)
• Reverse primer (The other primer binds to the 3’ end of
the anti-parallel DNA strand)
• Heat resistance DNA polymerase(Taq polymerase) Works at
high temperature
• All types of deoxynucleotide triphosphates (dNTPs)
adenine, cytosine, guanine and thymine.
• PCR buffer
• Divalent cations (MgCl2)

8. The basic steps are:
• Denaturation (95 °C)Heat the reaction strongly to
separate, or denature, the DNA strands. This
provides single-stranded template for the next
step.
• Annealing (55° C): Cool the reaction so the
primers can bind to their complementary
sequences on the single-stranded template DNA.
• Extension (72 °C): Raise the reaction
temperatures so Taq polymerase extends the
primers, synthesizing new strands of DNA.

9. How PCR Works• Protocol
– Put all reagents into a PCR tube
Melting or heat denaturation
Break the DNA ladder down the middle to create two
strands, a 5’ to 3’ strand and a 3’ to 5’ strand
Annealing
Bind each primer to its appropriate strand
• 5’ primer to the 5’ to 3’ strand
• 3’ primer to the 3’ to 5’ strand
Extending
Copy each strand
• DNA polymerase

10. How PCR Works
• Temperature Protocol
– Initial Melt: 95ºC for 2 minutes
– Melt: 95ºC for 30 seconds
– Anneal: 55ºC for 30 seconds
– Extend: 72ºC for 1 minute
– Final Extension: 72ºC for 6 minutes
– Hold: 4ºC
30-35
cycles

11. Why electrophoresis?
• To separate DNA
fragments from each
other
• To determine the sizes
of DNA fragments
• To determine the
presence or amount of
DNA
• To analyze restriction
digestion products

12. Introduction Of Agarose Gel
Electrophoresis
• Agarose gel electrophorresis is a method
to separate DNA or RNA molecules by size &
charge.
• This is achieved by moving negatively charged
nucleic acid molecules through an agarose
matrix with an electric field (electrophoresis).
• Shorter molecules move faster and migrate
faster than longer ones .

13. Principle of electrophoresis
• separates molecules from each other on the basis of
– size
– charge
– shape
• basis of separation depends on how the sample and gel
are prepared
• employs electromotive force to move molecules
through a porous gel

14. Material required for agarose gel
electrophoresis
 Electrophoresis chamber
 Agarose gel
 Gel casting tray
 Buffer
 Staining agent (dye)
 A comb
 DNA ladder
 Sample to be separate
 ETBR (ethidium bromide)

15. Electrophoresis Equipment

16. How Gel Electrophoresis of DNA Works

17. Agarose Gel Electrophoresis
Molecular Weight
Standard
(DNA of Known Sizes)
1 2 3 4 5 6 7 8 9 10
Samples of DNA
2000 bp
1000 bp
750 bp
Lanes:
5000 bp

18. There are many types of PCR
 Real-Time PCR (quantitative PCR or
qPCR)
 Reverse-Transcriptase (RT-PCR)
 Multiplex PCR
 Nested PCR
 High Fidelity PCR
 Fast PCR
 Hot Start PCR
 GC-Rich PCR
 Long-range PCR.
 Quantitative PCR
 Semi quantitative PCR
 Methylation specific PCR
 In situ PCR
 Long PCR
 Colony PCR
 Touch down PCR
 Band stab PCR Allele specific PCR
 Digital PCR
 Overlap Extension PCR
 Solid phase
 Inverse PCR
 Hot start PCR
 Differential display
 Ligation mediated PCR
 Methylation specific PCR

19. Real-Time PCR
• Real time PCR also k/a kinetic PCR, qPCR, qRT PCR
and RT-PCR, is a quantitative PCR method for the
determination of copy number of PCR templates
such as DNA or CDNA in a PCR reaction.
• Real-time PCR monitors the fluorescence emitted
during the reaction as an indicator of amplicon
production at each PCR cycle.
• Thus in Real-time PCR one can visually see the
progress of the reaction in "real time“

20. Reverse-Transcriptase (RT-PCR)
It is employed for amplification of RNA
molecules .
 RT-PCR is widely used in expression profiling,
to determine the expression of a gene or to
identify the sequence of an RNA transcript.

21. Multiplex PCR
• Multiplex PCR is a widespread molecular biology
technique for amplification of multiple targets in
a single PCR experiment.
• In a multiplexing assay, more than one target
sequence can be amplified by using multiple
primer pairs in a reaction mixture.
• Generally up to eight primer pairs c/b used in a
standard multiplex reaction, otherwise the yield
of some amplicons is reduced and not visible on
agarose gel.

22. Nested PCR
• Two pairs instead of one pair of PCR primers
are used to amplify a fragment.
• First pair amplifies a fragment similar to
standard PCR
• Second pairs bind inside the 1st PCR product
fragment allow amplification of 2d PCR
product which is shorter than the 1st one

23. Application of PCR
• Diagnosis of infectious disease
 Tuberculosis
 Hiv
• Detection of variation & mutations in gene
• DNA finger printing can multiply

24. Thank you