Usefulness of

Polymerase Chain Reaction

in disease diagnosis

History
The

Polymerase Chain Reaction (PCR) was not a

discovery, but rather an invention

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

Why to use PCR

PCR

can be used to make many copies of any

DNA that is supplied as a template

Starting

with one original copy an almost infinite

number of copies can be made using PCR
Amplified fragments of DNA can be sequenced,
cloned, probed or sized using electrophoresis

 Defective

genes can be amplified to

diagnose any number of illnesses

Amplified

fragments can act as genetic

fingerprints

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

Basics of DNA replication

Origin of Replication

5
5

5
5

The Replication Fork

5
3

5
3

3
5

Primase

Laging Strand
Okazaki
fragment

3
RNA
Primers

Single
strand
binding
proteins

DNA
Polymerase

5
3
Helicase

Leading Strand

5
3

Functions And Their Associated

Enzymes
Function
Enzyme
Melting

DNA

Helicase
SSB

Proteins
Topisomerase
Polymerizing

Providing

DNA

primer
Joining nicks

DNA Polymerase

Primase
Ligase

Temperature

PCR
100

Melting
94oC

o
Extension 94 C

Annealing
Primers
50oC

50

72oC

Time

5
3

3
5

5
3

5
3

5
5

5
5

30x

Melting

3
3

Temperature

100

Melting
94oC

PCR

50

Time

Temperature

100

Melting
94oC

PCR

50

Time
3

Heat
5

Temperature

100

Melting
94oC

50

PCR
Annealing
Primers
50oC

Melting
o
Extension 94 C
72oC

Time
3

5
5

Temperature

PCR
100

Melting
94oC

50

Melting
94oC
Extension
Annealing
72oC
Primers
50oC

Time
3

Heat
5

Heat
5
5

30x

Temperature

100

Melting
94oC

50

PCR
Annealing
Primers
50oC

Melting
o
Extension 94 C
72oC

Time
3

5
5

30x

Temperature

100

50

0
3

Melting
94oC

PCR
Annealing
Primers
50oC

Melting
o
Extension 94 C
72oC

Time
5

5
5

Heat
5

Heat
5

30x

Temperature

PCR
100

50

0
3

Annealing
Primers
50oC

Melting
o
Extension 94 C
72oC

Time
5

5
5

Melting
94oC

30x

Temperature

100

Melting
94oC

50

0
3

Annealing
Primers
50oC

Melting
o
Extension 94 C
72oC

Time

5
5

PCR

Fragments of
defined length

30x

DNA Between The Primers Doubles

With Each Thermal Cycle
Number
1
2

16

32

64

Cycles

More Cycles = More DNA

Theoretical Yield Of PCR

Theoretical yield = 2n x y
Where y = the starting
number of copies and
n = the number of thermal cycles
If you start with 100 copies, at the end of 30
cycles?

2n x y
=230 x 100
=1,073,741,824 x 100
=107,374,182,400

How The Functions Of Replication

Are Achieved During PCR
Function

Melting

PCR

DNA
Polymerizing
DNA
Providing primer

Heat

Joining

N/A as

nicks

Taq

DNA
Polymerase

Primers

are
added to the
reaction mix

short

fragments are

Components of a PCR Reaction

Buffer

(containing Mg++ Cl ++)

Template DNA
2 Primers that flank the fragment of
DNA to be amplified
dNTPs
Taq DNA Polymerase (or another
thermally stable DNA polymerase)

Templates for PCR

Dried

blood
Semen stains
Vaginal swabs
Single hair
Fingernail scrapings
Insects in Amber
Egyptian mummies
Buccal Swab
Toothbrushes

Heat-stable polymerase

Thermus aquaticus:

Preparation of master mix

10

X PCR Buffer with MgCl2

Primers F
Primers R
dNTP (building blocks of DNA)
Taq polymerase
Template DNA (will vary for each sample)
Make up the volume with DW

 The

reactions are carried out in thin walled

PCR tubes in a Thermal cycler

The amplified products

are
analysed
on
agarose
gels
and
visualized
on
Gel
documentation systems

Advantages of PCR
Quick
Reliable
Sensitive
Relatively
Specific

easy

PCR Applications in Pathology.

This technique is elegant and sensitive and has been

adopted quickly.
Diagnosis of infections is limited by the supply of
appropriate material for culture, protein analysis or
microscopy.
PCR has led to a better understanding of the pathobiology
of malignancy, allowing the analysis of mutations in
oncogenes and tumour suppressor genes.
PCR's greatest versatility is that it allows the examination
of formalin fixed paraffin wax embedded tissue.
PCR has brought significant progress in forensic pathology.

Disadvantage of PCR

Need for equipment

Taq polymerase is expensive
Contamination
False reactions
Internal control
Cross-reaction
Enrichment steps in (contaminated) samples
Skill building needed
Unspecific amplification

rtPCR

Nested primers

Nested primers

Multiplex-PCR
Multiple

primers are used in the same reaction mixture.

Simultaneously many genes can be targeted.
Produce amplicons of varying sizes that are specific to
different DNA sequences.

M
4

2
16sdys

paU

500bp
200bp

549bp

alr

361bp

sip
nuc

181bp

Hot start PCR.

It

reduces non-specific amplification

during the initial set up stages of the PCR.
The enzyme gets activated at the optimum
temperature.
It inhibits the polymerases activity at
ambient temperature, either by the binding
of an antibody.

Real Time PCR

Detection

and quantification of fluorescent reporter

the signal of which increases in direct proportion to
the amount of PCR product in a reaction.
Does not only measure the amount of end product
but its production in real time

 The

identity of the product can further be

analysed by direct sequencing
gene <1..887 /gene="ompH" CDS <1..887

/gene="ompH" /codon_start=3 /product="outer

membrane protein" /protein_id="AAT37506.1"
/db_xref="GI:47681474" /"
1 gcgtttcatt caaagcatct catgatttag gcgaaggctt aagcgcatta gcttatacag
61 aacttcgttt tagtaaaaat gtacccgtgc aagtaaaaga ccaacaaggt gaagtagtac
121 gtgagtatga ggttgagaaa cttggtaaca atgttcacgt aaaacgtctt tatgcgggtt
181 tcgcgtatga aggtttaggt acattaacat tcggtaacca attaactatc ggtgatgatg
241 ttggtctatc tgactatacc tatttcaaca gtggtattaa taacctcctt tctagcggtg
301 aaaaagcaat taactttaaa tctgcagaat tcaatggttt cacatttggt ggtgcgtatg
361 tcttctctgc tgatgctgac aaacaagcat tacgtgatgg tcgcggtttc gttgtagcag
421 gtttatacaa cagaaaaatg ggtgatgttg gttttgcatt cgaagccggt tatagccaaa
481 aatatgtgaa acaagaagta gaacaagcac aagcaccaaa agtatttaaa gatgaaaaag
541 agaaagcttt catggtgggt gctgagttat catatgctgg tttagcgctt ggtgttgact
601 acgcacaatc taaagtgact aacgtagatg gtaaaaaacg tgctcttgaa gtgggtttaa
661 attatgacct taacgacaga gcgaaagttt acacagactt catctgggaa aaagaaggtc
721 ctaaaggtga tgttacaaga aaccgtactg tcgctgtagg ttttggttac aaacttcaca
781 aacaagtgga aacttttgtt gaagcagctt ggggtagaga gaaagactct gatggtgtaa
841 caacaaaaaa caacgtagta ggtacaggtt tacgcgtaca cttctaattt ttgttagaat
901 ctgaaaaaag ccagtgttaa acactggctt tttattgggt tttatttgtt ttacttacaa
961 taaattagga ttttgaaagt cgttacgcgg tcat

References
Mullis,

Kary (1990). The unusual origin of

the polymerase chain reaction. Scientific
American 262 (4): 56-61, 64-65
Polymerase
chain
reaction
(
http://en.wikipedia.org/wiki/Polymerase_ch
ain_reaction
)