POLYMERASE CHAIN REACTION

Tapeshwar Yadav(Lecturer)BMLT, DNHE, M.Sc. Medical Biochemistry

Tapeshwar Yadav(Lecturer)BMLT, DNHE, M.Sc. Medical Biochemistry

• The Polymerase Chain Reaction (PCR) was not a discovery, but rather an invention

• In vitro technique for generating large quantities of a specified DNA.

• Karry Mullis, the inventor of PCR in 1989, was awarded the 1993 Nobel Prize in Chemistry

• PCR work was first published (1985)using Klenow polymerase – unstable with heat

• New enzyme had to be added manually at each step• Maximum length 400bp• Great idea – not very practical

• First reports using DNA polymerase

from Thermus aquaticus (1988)• Taq-polymerase (Saiki et al, 1988) from

Yellow stone National Park hot springs• Developed automatic “thermocycler” programmable

heat block

Development….

Polymerase Chain Reaction (PCR)

• PCR is a technique which is used to amplify the number of copies of a specific region of DNA, in order to produce enough DNA to be adequately tested.

• Cell-free amplification for synthesizing multiple identical copies (billions) of any DNA of interest.

• Basic tool for the molecular biologist.• The purpose of a PCR is to make a huge number of

copies of a gene. As a result, it now becomes possible to analyze and characterize DNA fragments found in minute quantities in places like a drop of blood at a crime scene or a cell from an extinct dinosaur.

• Like Xerox machine for gene copying.

standard tube, volume, costevaporation & heat transfer concerns

thin walled tube, volume, cost evaporation & heat transfer concerns

standard tube, volume, costevaporation & heat transfer concerns

thin walled tube, volume, cost evaporation & heat transfer concerns

PCR Thermocycler

heated lidsadjustable ramping timessingle/multiple blocksgradient thermocycler blocks

heated lidsadjustable ramping timessingle/multiple blocksgradient thermocycler blocks

ThermocyclersThermocyclers

What all PCR Can Do ?

• 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

• Genes from pathogens can be amplified to identify them (i.e., HIV, Vibrio sp., Salmonella sp. etc.)

• Amplified fragments can act as genetic fingerprints

PROCEDURE …..

PCR Reagents• 1X Buffer

– 10mM Tris-HCl, 50mM KCl• MgCl2

– 1mM - 4mM (1.5mM)• dNTPs

– 200μM• Primers

– 100nM-1μM, 200nm (or less) for real time analysis• DNA polymerase

– Taq DNA polymerase is thermostable– 1-4 Units (1 unit)

• DNA– 10pg-1μg (20ng)

MgCl2 (mM) 1.5 2 3 4 5

Magnesium Chloride (MgCl2 - usually 0.5-5.0mM)

Magnesium ions have a variety of effectsMg2+ acts as cofactor for Taq polymerase

Required for Taq to function

Mg2+ binds DNA - affects primer/template interactions

Mg2+ influences the ability of Taq pol to interact with primer/template sequences

More magnesium leads to less stringency in binding

Different types of buffers

Directional SynthesisDirectional Synthesis

Steps:1.Denaturation (Separation):- by heating at 95’C for 15 sec to 2 min.

2. Annealing (Priming):- primera are annealed by cooling to 50’C for 0.5 to 2 min.

3. Amplification (Polymerisation):-DNA strands are synthesized by Taq polymerase 72’C for 30 sec. in presence of dNTPS.

Steps:1.Denaturation (Separation):- by heating at 95’C for 15 sec to 2 min.

2. Annealing (Priming):- primera are annealed by cooling to 50’C for 0.5 to 2 min.

3. Amplification (Polymerisation):-DNA strands are synthesized by Taq polymerase 72’C for 30 sec. in presence of dNTPS.

Sources of DNA Polymerase: In the original technique of PCR, Klenow fragments of E.coli DNA polymerase was used.

This enzymes gets denatured at higher temp. therefore fresh enzyme had to be added each cycle.

Therefore introduction of Taq DNA Polymerase (Lawyer 1989) from thermophilic bacterium, Thermus aquaticus.

Taq DNA Polymerase is heat resistant, hence it is not necessary to freshly add this enzyme for each cycle to PCR.

“Xeroxing” DNA

1 copy

Cyc

le 1

PLU

S d

NTP

s, b

uffe

r,

salts

,Taq

pol

,

prim

ers

Cycle 35

n36 = 68,719,476,736 copies in ~ 2 hrs

2 copies

Cycle 2

4 copies

Cycle 3

8 copies

A simple thermocycling protocolA simple thermocycling protocol

annealingannealing

94ºC94ºC 94ºC94ºC

55ºC55ºC

72ºC72ºC

4ºC4ºC

3 min3 min 1 min1 min

45 sec45 sec

1 min1 min

∞ hold∞ hold

Initial denaturation of DNAInitial denaturation of DNA

1X1X 35X35X 1X1X

extensionextension

denaturationdenaturation

Step 1:DenaturationdsDNA to ssDNA

Step 2:AnnealingPrimers onto template

Step 3:ExtensiondNTPs extend 2nd strand

Step 1:DenaturationdsDNA to ssDNA

Step 2:AnnealingPrimers onto template

Step 3:ExtensiondNTPs extend 2nd strand

Vierstraete 1999

extension products in one cycle serve as template in the nextextension products in one cycle serve as template in the next

Polymerase Chain ReactionPolymerase Chain Reaction

Typical PCR Temps/TimesTypical PCR Temps/Times

holdhold4o C or 10 mM EDTA

4o C or 10 mM EDTA

Stop reactionStop reaction

5 – 10 min

5 – 10 min

70o – 75o C70o – 75o CFinal extension

Final extension

0.5 – 2 min

0.5 – 2 min

70o – 75o C70o – 75o CPrimer extension

Primer extension

0.5 – 1 min

0.5 – 1 min

45o – 65o C45o – 65o CPrimer annealing

Primer annealing

0.5 – 1 min

0.5 – 1 min

90o – 95o C90o – 95o CDenatureDenature

1 – 3 min

1 – 3 min

90o – 95o C90o – 95o CInitial denaturation

Initial denaturation

25 – 40 cycles

25 – 40 cycles

Initiation - Forming the Replication Eye

3’ 5’

3’5’5’

5’

3’

3’

Origin of Replication

5’

3’

3’

5’

5’3’

5’

5’

5’

3’

3’3’

Leading StrandLeading Strand

Laging StrandLaging Strand

3’

5’3’

5’

Extension - The Replication Fork5’

5’5’3’

3’

5’3’3’

5’

Single strand binding proteins

DNA Polymerase

Okazaki fragment

RNA Primers

Primase

5’3’

5’

Helicase

How are the functions of replication achieved during PCR ???

. N/A as fragments are short

Joining nicks

. . Taq Polymerase Polymerizing DNA

. . Primers added to the reaction mix

Providing primer

PCRFunction

. . Heat Melting DNA

ENZYMES

• Helicase•SSB proteins•Topoisomerase

•DNA pol

•Primase

•Ligase

PCRMelting

94 oC

Tem

pera

ture

100

0

50

T i m e

5’3’

3’5’

PCRMelting

94 oC

Tem

pera

ture

100

0

50

T i m e

3’5’

5’3’

Heat

PCRMelting

94 oCAnnealing

Primers50 oC

Extension72 oC

Tem

pera

ture

100

0

50

T i m e

3’5’

5’3’5’

5’

Melting94 oC

PCRMelting

94 oCMelting

94 oCAnnealing

Primers50 oC

Extension72 oC

Tem

pera

ture

100

0

50

T i m e

30x

3’5’

5’3’

Heat

Heat

5’

5’

5’

PCRMelting

94 oCMelting

94 oCAnnealing

Primers50 oC

Extension72 oC

Tem

pera

ture

100

0

50

T i m e

30x

3’5’

5’3’5’

5’

5’

5’

5’

5’

PCRMelting

94 oCMelting

94 oCAnnealing

Primers50 oC

Extension72 oC

Tem

pera

ture

100

0

50

T i m e

30x

3’5’

5’3’ 5’

5’5’

5’

5’

5’

Heat

Heat

PCRMelting

94 oCMelting

94 oCAnnealing

Primers50 oC

Extension72 oC

Tem

pera

ture

100

0

50

T i m e

30x

3’5’

5’3’ 5’

5’5’

5’

5’

5’

5’

5’

5’

5’

Fragments of defined length

PCRMelting

94 oCMelting

94 oCAnnealing

Primers50 oC

Extension72 oC

Tem

pera

ture

100

0

50

T i m e

30x

3’5’

5’3’ 5’

5’5’

5’

5’

5’

5’

5’

5’

5’

More Cycles = More DNA

Number of cycles 0 10 15 20 25 30

SizeMarker

PCR Optimisation 1: Buffers• Most buffers have only KCl (50mM) and Tris

(10mM)– Concentrations of these can be altered– KCl facilitates primer binding but

concentrations higher than 50mM inhibit Taq

• DMSO, BSA, gelatin, glycerol, Tween-20, Nonidet P-40, Triton X-100 can be added to aid in the PCR reaction– Enhance specificity, but also can be inhibitory

• Pre-mixed buffers are available

PCR Optimisation 2: MgCl2• MgCl2: required for primer binding

– MgCl2 affects primer binding, Tm of template DNA,

product- and primer-template associations, product

specificity, enzyme activity and fidelity

– dNTPs, primers and template chelate and sequester the

Mg ion, therefore concentration should be higher than

dNTPs (as these are the most concentrated)

– Excess magnesium gives non-specific binding

– Too little magnesium gives reduced yield

PCR Optimisation 3: Primer Design• Specific to sequence of interest

– Length 18-30 nucleotides• Annealing temperature 50oC-70oC

– Ideally 58oC-63oC• 3’ end critical (new strand extends from here)

• 3’ complementarity:– <3-4 bases similar to other primer regions

PCR Optimisation 4: Cycling Conditions

• Denaturation: – Some Taq polymerases require initial denaturation (hot

start)

• Annealing temperature: – ~ 5oC less than Tm of primers– Tm = 4(G + C) + 2(A + T)oC (or use of primer software)– Decrease in annealing temperature result in non-

specific binding– Increase in annealing temperature result in reduced

yield

PCR Optimisation 5: Cycle Number• 25-40 cycles• Half-life of Taq is

30 minutes at 95oC

• Therefore if you use more than 30 cycles at denaturation times of 1 minute, the Taq will not be very efficient at this point

Theoretical yield = 2n

ie. cycle 1 = 2, cycle 2 = 4, cycle 3 = 8, etc

eg. if you start with 100 copies after 30 cycles you will have 107, 374, 182, 400 copies

PRIME

• The PRIME is a good tool for the design of primers for PCR and sequencing– For PCR primer pair selection, you can choose a target range of

the template sequence to be amplified

• In selecting appropriate primers, PRIME allows you to specify a variety of constraints on the primer and amplified product sequences. – upper and lower limits for primer and product melting temperatures – a range of acceptable primer sizes – a range of acceptable product sizes. – required bases at the 3' end of the primer (3' clamp) – maximum difference in melting temperatures between a pair of PCR primers

PC Software• There are a number of (expensive) dedicated PCR

primers design programs for personal computers that have “special features” such as nested and multiplex PCR :– Oligo (Molecular Biology Insights, Inc.)– Primer Premier (Premier Biosoft)

• Many of the comprehensive MolBio. programs also have PCR features

– Mac Vector– OMIGA– Vector NTI– Gene Tool

Primer Problems

• primers should flank the sequence of interest

• primer sequences should be unique

• primers that match multiple sequences will give multiple products

• repeated sequences can be amplified - but only if unique flanking regions can be found where primers can bind

Variations of the PCR• Colony PCR• Nested PCR• Multiplex PCR• AFLP PCR• Hot Start PCR• In Situ PCR• Inverse PCR• Asymmetric PCR• Long PCR• Long Accurate PCR• Reverse Transcriptase PCR• Allele specific PCR• Real time PCR

Types of PCR

Long PCR: Used to amplify DNA over the entire length up to 25kb of genomic DNA

segments cloned.

Nested PCR: Involves two consecutive PCR reactions of 25 cycles. The first PCR uses primers external to the sequence of interest. The second PCR uses the product of the first PCR in conjunction with one or more nested primers to amplify the sequence within the region flanked by the initial set of primers.

Inverse PCR: Used to amplify DNA of unknown sequence that is adjacent to known DNA sequence.

Quantitative PCR: Product amplification w r t time, which is compared with a standard DNA.

Hot start PCR: Used to optimize the yield of the desired amplified product in PCR and simultaneously to suppress nonspecific amplification.

Colony PCRColony PCR- the screening of bacterial (E.Coli) or yeast clones for

correct ligation or plasmid products.

Pick a bacterial colony with an autoclaved toothpick, swirl it into 25 μl of TE autoclaved dH2O in an microfuge tube.

Heat the mix in a boiling water bath (90-100C) for 2 minutes

Spin sample for 2 minutes high speed in centrifuge.

Transfer 20 μl of the supernatant into a new microfuge tube

Take 1-2 μl of the supernatant as template in a 25 μl PCR standard PCR reaction.

Hot Start PCR• This is a technique that reduces non-specific amplification

during the initial set up stages of the PCR

• The technique may be performed manually by heating the reaction components to the melting temperature (e.g., 95°C) before adding the polymerase

• DNA Polymerase- Eubacterial type I DNA polymerase, Pfu

• These thermophilic DNA polymerases show a very small polymerase activity at room temperature.

Nested PCR

• Two pairs (instead of one pair) of PCR primers are used to

amplify a fragment.

• First pair -amplify a fragment similar to a standard PCR.

Second pair of primers-nested primers (as they lie / are

nested within the first fragment) bind inside the first PCR

product fragment to allow amplification of a second PCR

product which is shorter than the first one.

• Advantage- Very low probability of nonspecific amplification

Multiplex PCR

•  Multiplex PCR is a variant of PCR which enabling simultaneous amplification of many targets of interest in one reaction by using more than one pair of primers.

Inverse PCR• Inverse PCR (Ochman et al., 1988) uses standard PCR

(polymerase chain reaction)- primers oriented in the reverse direction of the usual orientation.

• The template for the reverse primers is a restriction fragment that has been selfligated

• Inverse PCR functions to clone sequences flanking a known sequence. Flanking DNA sequences are digested and then ligated to generate circular DNA.

Application• Amplification and identification of flanking sequences such

as transposable elements, and the identification of genomic inserts.

Long PCR

• Extended or longer than standard PCR, meaning over 5 kilobases (frequently over 10 kb).

• Long PCR is useful only if it is accurate. Thus, special mixtures of proficient polymerases along with accurate polymerases such as Pfu are often mixed together.

• Application- to clone large genes

Reverse Transcriptase PCR

• Based on the process of reverse transcription, which reverse transcribes RNA into DNA and was initially isolated from retroviruses.

• First step of RT-PCR - "first strand reaction“-Synthesis of cDNA using oligo dT primers (37°C) 1 hr.

• “Second strand reaction“-Digestion of cDNA:RNA hybrid (RNaseH)-Standard PCR with DNA oligo primers.

• Allows the detection of even rare or low copy mRNA sequences by amplifying its complementary DNA.

Why real time PCR ?

• QUANTITATION OF mRNA

– northern blotting– ribonuclease protection assay– in situ hybridization– RT-PCR

• most sensitive

• can discriminate closely related mRNAs

• technically simple

• but difficult to get truly quantitative results using conventional PCR

Real-Time PCRReal-Time PCR

Real-time PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon production at each PCR cycle (in real time) as opposed to the endpoint detection

• Traditional PCR has advanced from detection at

the end-point of the reaction to detection while the

reaction is occurring (Real-Time).

• Real-time PCR uses a fluorescent reporter signal

to measure the amount of amplicon as it is

generated. This kinetic PCR allows for data

collection after each cycle of PCR instead of only

at the end of the 20 to 40 cycles.

Real-time PCR advantagesReal-time PCR advantages

* amplification can be monitored real-time

* no post-PCR processing of products (high throughput, low contamination risk)

* ultra-rapid cycling (30 minutes to 2 hours)

* wider dynamic range of up to 1010-fold

* requirement of 1000-fold less RNA than conventional assays

(6 picogram = one diploid genome equivalent)

* detection is capable down to a two-fold change

* confirmation of specific amplification by melting curve analysis

* most specific, sensitive and reproducible

* not much more expensive than conventional PCR(except equipment cost)

Real-time PCR disadvantagesReal-time PCR disadvantages

* Not ideal for multiplexing

* setting up requires high technical skill and support

* high equipment cost

* intra- and inter-assay variation

* RNA liability

* DNA contamination (in mRNA analysis)

Applications of PCRApplications of PCR

• Classification of organisms

• Genotyping• Molecular archaeology

• Mutagenesis• Mutation detection

• Sequencing• Cancer research

• Detection of pathogens

• DNA fingerprinting

• Drug discovery• Genetic matching• Genetic engineering

• Pre-natal diagnosis

1.PCR in clinical diagnosis:

specificity & sensitivity of PCR is highly useful for the diagnosis of various diseases in humans.

Eg- Inherited disorders (genetic diseases), Viral diseases, Bacterial diseases etc

i) Prenatal diagnosis of inherited diseases

ii) Diagnosis of retroviral infections

iii) Diagnosis of bacterial infections

iv) Diagnosis of cancers

v) Sex determination of embryos

2. PCR in DNA sequencing

3. PCR in forensic medicine

4. PCR in comparative studies of genomes.

PCR Virtues

• High sensitivity• Can detect and quantify specific events• Higher stability of DNA permits analysis of food

samples.• Quantitative and qualitative

Thank you

THANK YOU