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PCR

Polymerase Chain
Reaction

What is PCR?
PCR is an exponentially progressing synthesis
of the defined target DNA sequences in vitro.
It was invented in 1983 by Dr. Kary Mullis, for
which he received the Nobel Prize in Chemistry in
1993.

What is PCR? :
Why Polymerase?
It is called polymerase because the only enzyme
used in this reaction is DNA polymerase.

What is PCR? :
Why Chain?
It is called chain because the products of the
first reaction become substrates of the
following one, and so on.

Polymerase Chain Reaction


(PCR)
PCR

is a means to amplify a particular piece of DNA

Amplify= making numerous copies of a segment of DNA

PCR

can make billions of copies of a target


sequence of DNA in a few hours

PCR

was invented in the 1984 as a way to make


numerous copies of DNA fragments in the laboratory

Its

applications are vast and PCR is now an integral


part of Molecular Biology

Key enzymes involved in DNA


Replication
DNA

Polymerase

DNA

Ligase

Primase
Helicase
Topoisomerase
Single

strand binding protein

DNA Replication vs. PCR


PCR

is a laboratory version of DNA


Replication in cells
The

laboratory version is commonly


called in vitro since it occurs in a test
tube while in vivo signifies occurring in
a living cell.

DNA Replication in Cells (in


vivo)

DNA replication is the copying of


DNA

It typically takes a cell just a few


hours to copy all of its DNA

DNA replication is semiconservative (i.e. one strand of the


DNA is used as the template for
the growth of a new DNA strand)

This process occurs with very few


errors (on average there is one
error per 1 billion nucleotides
copied)

More than a dozen enzymes and


proteins participate in DNA
replication

What is PCR? :
The Reaction Components

1) Target DNA - contains the sequence to be am

2) Pair of Primers - oligonucleotides that define


to be amplified.

3) dNTPs - deoxynucleotidetriphosphates: DNA


4) Thermostable DNA Polymerase enzyme that catalyzes the reaction
5) Mg++ ions - cofactor of the enzyme
6) Buffer solution maintains pH and
ionic strength of the reaction solution
suitable for the activity of the enzyme

Typical PCR mix


In a thin wall Eppendorf tube assemble the
following
PCR components
Amount
Template DNA (5-200 ng)
1 mM dNTPs (200 uM final)
10 X PCR buffer
25 mM MgCl2 (1.5 mM final)
20 uM forward primer (20 pmoles final)
20 uM reverse primer (20 pmoles final)
5 units/uL Taq DNA polymerase (1.5 units)
Water

variable
10 uL
5 uL
3 uL
1 uL
1 uL
0.3 uL
Variable

Final Volume

50 uL

PCR Reagents
1X

Buffer

10mM

Tris-HCl, 50mM KCl

MgCl2
1mM

- 4mM (1.5mM)

dNTPs

200M

Primers

100nM-1M,

DNA
Taq

200nm (or less) for real time analysis

polymerase

DNA polymerase is thermostable


1-4 Units (1 unit)

DNA

10pg-1g

(20ng)

Different types of buffers

Thermal Cycling
A

PCR machine controls


temperature

Typical

PCR go through three steps

Denaturation
Annealing
Extension

PCR Thermocycler

Denaturation
Heating

separates the
double stranded DNA
Denaturation

Slow cooling anneals


the two strands

Renaturation

Heat

Cool

Annealing
Two

primers are supplied in molar excess

They

bind to the complementary region

As

the DNA cools, they wedge between two template


strands

Optimal

temperature varies based on primer length etc.

Typical temperature from 40 to 60 C

Extension

DNA polymerase duplicates DNA


Optimal temperature 72C

PROCEDURE ..

PCR Amplification
Exponential Amplification of template DNA

Applications of PCR
Basic Research

Mutation screening
Drug discovery
Classification of organisms
Genotyping
Molecular Archaeology
Molecular Epidemiology
Molecular Ecology
Bioinformatics
Genomic cloning
Site-directed mutagenesis
Gene expression studies

Applied Research

Genetic matching
Detection of pathogens
Pre-natal diagnosis
DNA fingerprinting
Gene therapy

Applications of PCR
Molecular Identification
Sequencing

Genetic Engineeri

Site-directed mutagen
Molecular Archaeology Bioinformatics
Molecular Epidemiology
Genomic cloning Gene expression stud
Molecular Ecology
Human Genome Project
DNA fingerprinting
Classification of organisms
Genotyping
Pre-natal diagnosis
Mutation screening
Drug discovery
Genetic matching
Detection of pathogens

MOLECULAR

IDENTIFICATION:

Molecular Identification:

Detection of Unknown Mutations

Molecular Identification:

Detection Of Pathogens

Variations of the PCR

Thank you

THANK YOU