INTRODUCTION

● PCR is a technique that results in exponential

amplification of a selected region of a DNA molecule.

● PCR is widely held as one of the most important

inventions of the 20th century in molecular biology.

● The idea of PCR is credited to Kary Mullis, a research

scientist at California Biotech Company, Cetus, in 1983.

● For this work, Mullis received the Nobel Prize in

Chemistry jointly with Michael Smith in 1993.

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developed the PCR (polymerase chain reaction)

Kary Banks Mullis(Dec 28, 1944 - )

Nobel Prize in Chemistry 1993

1985 1986 1987 1988

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Kary Mullis received the Nobel Prize in Chemistry jointly with

Michael Smith in 1993.4

CONSTITUENTS OF PCR

REACTION

One or more molecules of target DNA

Two oligo-nucleotide primers(forward and reverse primers)

All the four (dNTPs)

Thermostable DNA polymerase

PCR Buffer to maintain pH

Divalent cation (Mg++).

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STEPS IN PCR REACTION

Denaturation of Double Stranded DNA (ds DNA)

Annealing of Primers to Single Stranded DNA template

Extension of primer or Synthesis of ds DNA.

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Denaturation of ds DNA

Convertion of the ds DNA molecule to ss DNA.

This reaction is usually performed at 94oC .

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Annealing of Primers to ss DNA

Template The base pairing of a single stranded primer to its

complementary region of the ss DNA molecule is known

as annealing.

The common choice of temperature range for this reaction

is 55-60oC.

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Extension of primers or Synthesis of ds DNA

The annealing of a primer provides a free 3’-OH

group for synthesis of ds DNA by thermostable

DNA polymerase using ss DNA as a template.

Extension is the synthesis of DNA by a thermostable

DNA polymerase using 3’-OH end of a primer.

It is done at 72oC, the optimal working temperature

for thermostable DNA polymerase.9

PCR procedure: cycle 1

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PCR procedure: cycle 2

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PCR procedure: cycle 3

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Temperature profile of PCR

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THEMOSTABLE DNA

POLYMERASES Originally thermostable polymerase was purified from the hot

spring bacterium Thermus aquaticus .

The thermophilic DNA polymerases catalyze template directed

synthesis of DNA using nucleotide triphosphates.

A primer having a free 3’-OH is required to initiate synthesis

and Mg++ ion is necessary.

These enzymes have maximum catalytic activity in the

temperature range of 75oC to 80oC.

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Thermostable DNA Polymerase

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Properties of Different

Thermostable DNA PolymerasesEnzyme Source M.W.(KDa) Optimum

temperature

(oc)

Exonuclease

activity

Fidelity Stability(Half

–Life)

Remarks

Taq DNA

polymeras

e(natural)

Thermus aquaticus 94 74 3-5

Low 40 min at

90oC

Use in routinePCR exp.

AmpliTaq Thermus

aquticus

- 75-80 None Low 21 min.at

97.5oC

Processivit

y is lower.

Tli DNA

polymerase(

Recombinan

t

Thermococc

us litoris

90 74 3-5 High 400min at

95oC

Primer

extension

and high

fidelity .

Pfu DNA

polymerase(

natural)

Pyrococcus

furiosus

90 75 3-5 High 240 min.at

95oC

High

fidelity and

primer

extension

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PRIMER DESIGNING PARAMETERS

Primer Length

Melting Temperature (Tm)

Primer Dimer

GC Content

Runs and Repeats

Distance Between Primers

Secondary Structures

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Primer Length

Optimal length of PCR primer is 18-30 nucleotides.

This length is sufficient for adequate specificity and short enough for the primer to bind to the template at the annealing temperature.

Shorter primers lead to amplification of nonspecific PCR products.

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Melting Temperature (Tm) Tm of a DNA molecule is defined as the temperature

at which one-half of the duplex DNA will dissociate to become single stranded.

Tm indicates the duplex stability.

The specificity of PCR depends strongly on the melting temperature of the primers.

Tm of primer hybridization can be calculated using various formulae:

(i) Tm =4 (G+C) + 2 (A+T) o C (commonly used)

(ii) Tm =22 +1.46 [2 x (G+C) +(A+T)]

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Primer Dimer

Primer dimers are generally two types:

(i) Self dimer: It is formed by intermolecular interaction between the two same primers, where the primer is homologous itself ;

(ii) Cross dimer : It is formed by intermolecular interaction between complementary regions of two different primers, i. e., sense and antisense.

The formation of primer dimers prevents the hybridization of primers to the template DNA, there by reducing the product yield.

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GC Content

The GC content of a primer should be 40-60%.

The presence of G or C bases within the last five bases

from the 3’-end of a primer helps to promote specific

binding at 3’-end due to the stronger bonding of G and C

bases.

More than three G or C should be avoided in last 5 bases

at the 3’-end of a primer.

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Runs and Repeats

Primer should lack stretches of polynucleotide sequences,

i.e., runs (e.g., poly dA) or repeating motifs, because these

can hybridize at wrong places on the template.

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Distance between Primers

Theoretically, the least distance between the primers on

template DNA should be 150 bp and utmost 10 kbp.

Typically, yield is reduced when the primers extend from

each other beyond 3 kbp approx.

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Secondary Structure

Presence of the secondary structures produced by

intermolecular or intramolecular interactions can lead to

poor or no yield of the product.

These adversely affect primer template annealing and thus

the amplification.

These greatly reduce the availability of the primers to the

reaction.

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CYCLE NUMBER

The number of amplification cycles necessary to produce

a band visible on a gel depends largely on the starting

concentration of the template DNA.

It is recommended that to amplify 50 target molecules 40-

45 cycles are needed,while 25-30 cycles are required to

amplify 3 x 105 molecules to the same concentration. This

non- proportionality is called Plateau effect which is the

decrease in the exponential rate of product accumulation

in late stages of a PCR.

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PCR PRODUCT YIELDThe predicted yield of PCR product can be calculated by a

simple ‘invested equation,

PCR product yield =(input target amount) x (1 + %

efficiency) x number of cycles

Thus , ~26 cycles are required to produce 1 mg of PCR

product from 1pg of a target sequence with an efficiency

value of 70%,i.e.,

1 mg PCR product =(1 pg target ) x 1+0.7) x26

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Amplification of target gene

30 cycles --- 1 billion copies in theory

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VERIFICATION OF PCR PRODUCTS After PCR reaction is completed, the agarose gel

electrophoresis is performed to determine the following:

(i)Whether or Not a Product is formed

(ii)Whether or Not the Product Formed is Right Size

(iii)Whether or Not a Single Band of Right size is formed

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FACTORS AFFECTING PCR AMPLIFICATION

Buffer Composition

Quality of Primers and their Concentrations

Nucleotides Concentration

Primer Annealing temperature

Choice of Polymerases for PCR

Cycle Number

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Buffer Composition

Buffer for PCR reaction generally contains:

(i) Tris-HCl 10-15mM pH 8.3

(ii) KCl 50mM

(iii) MgCl2 1.5mM

(iv) Primers (forward and reverse) 0.2-1 µM each

(v) dNTPs 50-200 µM each

(vi)Gelatin or BSA up to 100µg/ml and Non-ionic

detergents such as Tween-20/Triton X-100

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Quality of Primers and Their Concentrations Good primer design is indispensable for successful

reaction.

The primers anneal to the complementary sequences on the template DNA and thereby determine the boundaries of the amplified product.

Primer concentration is also important .

It should not go above 1 µM except for degenerate primers.

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Nucleotides Concentration

Nucleotides concentration should not exceed 50 µM each.

Long products may need higher concentration.

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Primer Annealing Temperature

Ta of the PCR reaction is determined on the basis of Tm

of primer.

Too high Ta will produce insufficient primer-template hybridization resulting in low PCR product yield.

If the Ta is too low, it may lead to the formation of nonspecific products because of high number of mismatches.

Ta can be calculated by this formula:

Ta =0.3 x Tm(primer) + 0.7 Tm (product) - 14.9

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Choice of Polymerases for PCR

The enzymes used for polymerization also affect the PCR amplification.

The polymerases used in synthesis of DNA should be thermostable up to the temperature 94oC.

The polymerases lacking 3,-5, exonuclease activity generally have higher error rates than the polymerases with exonucleases activity.

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Cycle Number

The number of amplification cycles necessary to produce

a band visible on a gel depends largely on starting

concentration of the target DNA.

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