The document discusses the polymerase chain reaction (PCR) process. It describes how PCR was invented to amplify specific segments of DNA using primers and thermocycling. The key steps of PCR include denaturation to separate DNA strands, annealing of primers, and extension of new strands by DNA polymerase. PCR is a simple yet powerful technique with many applications such as genotyping, sequencing, and mutation detection. It allows targeted amplification of DNA for analysis.

1. ©2000 Timothy G. Standish
Polymerase Chain Reaction
Mrs. S.Kousalya M.Sc., M.Phil.,
Assistant Professor
Department of Microbiology
VIAAS, Sankagiri, Salem.
DEPARTMENT OF MICROBIOLOGY
VIVEKANANDAARTS AND SCIENCE COLLEGE FOR
WOMEN SANKAGIRI

2. ©2000 Timothy G. Standish
History
The Polymerase Chain Reaction (PCR) was not a
discovery, but rather an invention
After DNA extraction, the scientist want to study a
specific part of a gene to do sequencing
A special DNA polymerase (Taq) is used to make
many copies of a short length of DNA (100-10,000
bp) defined by primers
Kary Mullis, the inventor of PCR, was awarded the
1993 Nobel Prize in Chemistry

3. ©2000 Timothy G. Standish
PRIMER
In a PCR reaction you need two primers to
amplify the target sequence:
Forward primer-which have the same sequence of
forward DNA strand and bind to the
complementary reverse strand.
Reverse primer-which have the same sequence of
reverse DNA strand and bind to the
complementary forward strand
Amplified fragments can act as genetic fingerprints

4. ©2000 Timothy G. Standish
How PCR Works
PCR is an artificial way of doing DNA
replication
Instead of replicating all the DNA
present, only a small segment is
replicated, but this small segment is
replicated many times
As in replication, PCR involves:
–Melting DNA
–Priming
–Polymerization

5. ©2000 Timothy G. Standish
• PCR proceeds in THREE steps involved
(Temperature)
Denaturation: (95⁰C)
• The double-stranded template DNA is
denatured by heating, typically to 95°C, to
separate the double stranded DNA.

6. ©2000 Timothy G. Standish
Annealing: (50-65⁰C)
The reaction is rapidly cooled to an
annealing temperature to allow the
oligonucleotide primers to hybridize to the
template.

7. ©2000 Timothy G. Standish
Extension(72°C)
The reaction is heated to a temperature,
typically 72°C for efficient DNA synthesis
by the thermostable DNA polymerase.

8. ©2000 Timothy G. Standish
Components of a PCR
Reaction
Buffer (containing Mg++)
Template DNA
2 Primers that flank the fragment of
DNA to be amplified
dNTPs
Taq DNA Polymerase (or another
thermally stable DNA polymerase)

9. ©2000 Timothy G. Standish
PCR Advantages
Simplicity, easier methodology, sensitive,
extensively validated standard operating
procedure and availability of reagents and
equipment

10. ©2000 Timothy G. Standish
PCR Application
Genotyping.
RT-PCR.
Cloning.
Mutation detection.
Sequencing.
Microarrays.

11. ©2000 Timothy G. Standish
Disadvantages
Some time non specific binding of primer to
other sequence on the template DNA.

12. ©2000 Timothy G. Standish