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Introduction

to the

PolymeraseChain ReactionAmplification

(PCR)

The Invention of PCR

• Invented by Kary

Mullis in 1983

• Awarded Nobel

Prize for Chemistry

in 1993

You can read his Nobel lecture here:http://nobelprize.org/nobel_prizes/chemistry/laureates/199

3/mullis-lecture.html

What is PCR?

•  Polymerase Chain Reaction = repeated, sequence-specific amplification of nucleic acid (usually

DNA).

Why is it the ‘in’ technique & why is it so useful? 

•  It’s quick (can only take a few hours – result).

•  It’s sequence-specific – the ends of the amplified

piece of nucleic acid are define by the design of the

primers.

•  By molecular biology standards it’s quite cheap. 

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• PCR is a Key technique in the Forensic analysis of DNA

• Genetic profiling in humans based on the analysis of short

tandem repeat sequences –  STR’s 

ATCG¦ATCG¦ATCG¦ATCG¦ATCG¦ATCG¦ATCG¦ATCG = 8 repeats

ATCG¦ATCG¦ATCG¦ATCG¦ATCG¦ATCG = 6 repeats

Allele  – 8 and Allele – 6

Position of an STR on a chromosome – locus. Different STRs

found at different loci.

Individuals can be homozygous or heterozygous for a particular 

STR 

• Each person has a particular set of STR alleles of different loci –  

analyse enough and this becomes a ‘unique’ profile within a

 population – used to link individuals to biological material found at

crimes scenes….. 

•PCR is used to specifically amplify STR loci therefore identify

allele profiles… 

How Does It Work?

•  PCR mimics cellular DNA replication in 

vitro (inglass).

•  Repeats the equivalent of 30 – 40 rounds of 

semi- conservative DNA replication in a few

hours.

• Uses DNA polymerases to add

deoxyribonucleotides to a growing DNA

strand complementary to the target sequence• DNA replication always proceeds in a 5’ to 3’

direction.

HOW DO WE MIMIC DNA REPLICATION

IN VI TRO ?

•  Supply all components and incubate at 37°C ? –  

NO

•  Generally need to know some details of the DNA

sequence of interest - Why?

•  Allows the design of oligonuleotide primers which

are needed for the DNA polymerase to initiate

polymerisation – i.e. DNA synthesis.

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The different steps of PCR 

The cycling reactions :

There are three major steps in a PCR, which are repeated for 20 to 40 cycles.

This is done on an automated Thermo Cycler, which can heat and cool the

reaction tubes in a very short time.

Denaturation at around 94°C :

During the denaturation, the double strand melts open to single stranded DNA,

all enzymatic reactions stop (for example the extension from a previous cycle).

Annealing at around 54°C :

Hydrogen bonds are constantly formed and broken between the single stranded

 primer and the single stranded template. If the primers exactly fit the template,

the hydrogen bonds are so strong that the primer stays attached

Extension at around 72°C :The bases (complementary to the template) are coupled to the primer on the 3'

side (the polymerase adds dNTP's from 5' to 3', reading the template from 3' to

5' side, bases are added complementary to the template)

Choice of cycling parameters: determined empirically.

Typically:

94°C for 5 min – denature the target

94°C for 1 min) denaturation

55-60°C for 1 min) x35 primer annealing*

72°C for 1-5 min) primer extension

Plus:

72°C for 10 min to ensure amplicons (target amplified) are

complete

*annealing temp is varied 35-70 °C until optimised for

product yield.

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The temperature profile of 

a PCR cycle is controlled by

the thermal cycler program 

which results in a near 

exponential increase in PCR 

 product accumulation for 

about the first 30 cycles.

For a PCR reaction we need to supply:

• Template DNA (Target)

• Downstream Oligonucleotide Primer

• Upstream Oligonucleotide Primer

• Taq DNA Polymerase

• Reaction Buffer (x10 NH4 buffer)

• Cofactor - MgCl2

• Nuclease Free Water

• Nulease Free Light Mineral Oil (sometimes)

• Deoyribonucleotide triphosphates = dNTPs

Oligonucleotide Primer Design

Criteria:

•  Require a primer for each end of the two strands

that will define the ‘ends’ of the molecule

synthesised.

•  The primers must have their 3’OH ends pointing

towards each other such that they flank the

sequence to be amplified –  primers are ‘used up’ in

the process and become part of the product.

•  They are normally sequence specific and

therefore target specific.

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Primer Considerations:

•  Ideally should only bind to the sequence of 

interest… 

•  Should be ~ 18-40 bases long… 

•  Should have a ‘high’ G+C content… 

• Primer needs to be exact match with target – DNA

polymerase needs a fully matched primer template

sequence to begin polymerisation.

To mimic cellular DNA replication, temperature

changes are used to:

•  Denature the target – make the double

stranded DNA molecule single-stranded

•  Anneal (stick) the primers to their

complementary sequences

•  Extend the primers by enzymatic addition of 

dNTPs to the end of the primers (3’OH) to

‘copy’ the strands of the duplex 

•  All things being optimal, it results in a

roughly geometric increase in PCR 

amplicon (the bit you are amplifying)

This assumes that you:

•  Start with a single target molecule

•  Carry out e.g. 30 rounds of the cycle

•  You should end up with – 1 073 741 824 = 230 

molecules of amplicon

•  X = 2n (where n=no. molecules after n cycles)

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Exponential Amplification:

Cycle Number No. Product

Molecules

Expressed as an

Exponent

0 (start) 1 (initial target) 20

1 2 21

2

etc

4

etc

22

ect

10 1,024 210

This assumes that the following conditions hold true:

•  Start with 1 target molecule

•  An excess of primers & dntps are available

•  Over time buffer conditions do not change – pH etc.

•  Each step of the reaction has the same buffer & pH

requirements

•  100% priming etc occurs on every molecule at each

step

•  Product inhibition does not occuroccurs at ~1012

molecules / 100 µl of reaction

PCR target molecules

accumulate as a function

of cycle number. The

exponential phase lasts

for about 30 cycles under 

standard reactions

conditions.

The plateau phase results

from limiting amounts of 

enzyme and reduced

enzyme activity

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How are these quick sequential changes of temperature

achieved?

• Heats & Cools quickly:

• Block material – Silver, Teflon Coated Copper..

• Use of liquid medium – e.g. ethylene glycol –  

better uniform heating of reaction vessels

• Type and distribution of heating mechanism –  

usually an electrically heated coil

• Type of cooling mechanisms – water-cooled, fan… 

• Programmable

• Versatile reaction vessel capacity – 0.5ml, microtitre

plates… 

• Individual rows of receptacles can be heated – can try

lots of reaction temperatures in one ‘run’