Pcr

به نام خداPolymerase chain reaction

PCR by : Mahdi zarei

M.Sc. Student ,clinical

biochemistry

Ferdowsi university of mashhad

History:• By 1971 researchers in Khorana's project, concerned over their

yields of DNA, began looking at "repair synthesis" - an artificial system of primers and templates that allows DNA polymerase to copy segments of the gene they are synthesizing. Although similar to PCR in using repeated applications of DNA polymerase, the process they usually describe employs just a single primer-template complex, and therefore would not lead to the exponential amplification seen in PCR.

• Kary Mullis is generally credited with inventing PCR in 1983 while working for Cetus Corporation in Emeryville, California. While driving on Highway 128 from San Francisco to Mendocino, Mullis made an intellectual leap. He reasoned that by using two opposed primers, one complementary to the upper strand and the other to the lower, then performing multiple cycles of denaturation, annealing and polymerization he could exponentially amplify the piece of DNA between the primers.

PCR was invented in 1983 by ( Kary mullis ) & he received the Nobel Prize in chemistry in 1993, for his invention.

It revolutionized biological methods specially in molecular cloning in a way that it has became an inseparable & irreplaceable part of molecular investigations.

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DEVELOPMENT OF PCR

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

– not very practical

First reports using DNA polymerase from Thermus aquaticus (1988)

• Taq-polymerase (Saiki et al, 1988) from Yellowstone National Park hot springs

Development….

Thermostable Polymerases Polymerase T ½,

95oC Extension

Rate (nt/sec) Type of

ends Source

Taq pol 40 min 75 3’A T. aquaticus

Amplitaq (Stoffel

fragment)

80 min >50 3’A T. aquaticus

Vent* 400 min >80 95% blunt

Thermococcus litoralis

Pfu >120 min 60 Blunt Pyrococcus furiosus

Tth* (RT activity)

20 min >33 3’A T. thermophilus

*Have proof-reading functions and can generate products over 30 kbp

Automation of PCR

Developed automatic “thermocycler” programmable heat block…

• The early PCR experiments , researchers had to rely on a series of water baths to maintain the different temperatures required by the procedure . “cycling” involved manual transfer of samples from one water bath to another at specified times.

• In 1988, perkin-elmer introduced the thermal cycler , a revolutionary device that automatically and repetitively raised and lowered the temperature of the samples during PCR cycles . This allowed the PCR technique to be automated. Subsequent refinements of this device extended the flexibility and accuracy of pcr.

• While in some old machines the block is submerged in an oil bath to control temperature, in modern PCR machines a Peltier element is commonly used.

PCR Thermocycler

Stage of pcr Exponential amplification:

At every cycle, the amount of product is doubled (assuming 100% reaction efficiency). The reaction is very sensitive.

Leveling off (linear)stage: The reaction slows as the DNA

polymerase loses activity and as consumption of reagents such as dNTPs and primers causes them to become limiting.

Plateau: No more product accumulates due

to exhaustion of reagents and enzyme.

COMPONENT OF PCR REACTION&PCR PROCEDURE

Components of PCR Reaction

• Template DNA• Flanking Primers• Thermo-stable polymerase

• Taq Polymerase

• dNTP • (dATP, dTTP, dCTP, dGTP)

• PCR Buffer (mg++)• Thermocyler

Thermus aquaticus

primers

1. PCR primers should be 10-24 nucleotides in length.

2. The GC content should be 40%-60%.

3. The primer should not be self-complementary or complementaryto any other primer in the reaction mixture, to prevent primer-dimer and hairpin formation.

4. Melting temperatures of primer pairs should not differ by more than 5°C, so that the GC content and length must be chosen accordingly.

5. The melting and annealing temperatures of a primer are estimated as follows: if the primer is shorter than 20 nucleotides, the approximate melting temperature is calculated with the formula:

Tm = 4(G + C) + 2 (A + T)

6. The annealing temperature should be about 5°C lower than the melting temperature.

PCR Buffer Basic Components

20mM Tris-HCL pH 8.4 50mM KCl 1.5 mM MgCl2

Magnesium – Since Mg ions form complexes with dNTPs, primers and DNA templates, the optimal concentration of MgCl2 has to be selected for each experiment. Too few Mg2+ ions result in a low yield of PCR product, and too many increase the yield of non-specific products and promote mis incorporation.

Potential Additives Helix Destabilisers - useful when target DNA is high G/CWith NAs

of high (G+C) content. dimethyl sulphoxide (DMSO), dimethyl formamide (DMF), urea formamide 

Long Targets >1kb. Formamide and glycerol   Low concentration of template: Polyethylene glycol (PEG)

Temperature

Denaturation Trade off between denaturing DNA and not

denaturing Taq Polymerase Taq half-life 40min at 95 °, 10min at 97.5°

95° Annealing

Trade off between efficient annealling and specificity

2-5 ° below Tm Extension

Temperature optimum for Taq Polymerase 72 °

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

Detection of pcr productDetection Visualization

Agarose gel and/or polyacrylamide gel electrophoresis

-EtBr staining (UV transilluminator, image analyzer) -Southern blotting (hybridization with labeled probe) -Silver staining

Restriction endonuclease digestion

-Agarose or polyacrylamide gel -HPLC

Dot blots Hybridization with labeled probe

High-pressure liquid chromatography UV detection

ElectrochemiluminescenceVoltage-initiated chemical reaction/photon detection

Direct sequencingRadioactive or fluoescent-based DNA sequencing

Controls for PCR

Blank reaction

Controls for contamination

Contains all reagents except DNA template

Negative control reaction

Controls for specificity of the amplification reaction

Contains all reagents and a DNA template lacking the target sequence

Positive control reaction

Controls for sensitivity

Contains all reagents and a known target-containing DNA template

Interpretation of the PCR Results

The PCR product should be of the expected size.

No product should be present in the reagent blank.

Misprimes may occur due to non-specific hybridization of

primers.(pcr product present in the negative control)

Primer dimers may occur due to hybridization of primers

to each other.

Variations of the PCR

Hot Start PCR

It is a method for increasing specificity of PCR

reactions.

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

Ampliwax or antibody are used in hot start pcr

Nested PCR

It is a method for increasing specificity of PCR

reactions.

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 - 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

Touchdown PCR

It is a method for increasing specificity of PCR reactions.

Touchdown PCR uses a cycling program where the annealing

temperature is gradually reduced (e.g. 1-2°C /every second cycle).

The initial annealing temperature should be several degrees

above the estimated Tm of the primers. The annealing

temperature is then gradually decreased until it reaches the

calculated annealing temperature of the primers or some degrees

below. Amplification is then continued using this annealing

temperature.

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.

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.

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.

Real-time PCR

real-time polymerase chain reaction, also called

quantitative real time polymerase chain reaction

(qPCR) or kinetic polymerase chain reaction.

Real-time PCR detects and measures the

amplification target DNA as they are produced.

Unlike, conv. PCR, real-time PCR uses an

oligonucleotide probe labeled with fluorescent dyes

or an alternative chemistry, and a thermocycler

equipped with the ability to measure fluorescence.

Real time PCR in comparison with other technical methods

amplification can be monitored real-time no post-PCR processing of products

(high throughput, low contamination risk)

Less time to getting results No gel-based analysis at the end of the pcr reaction Computer based analysis of the cycle-fluorescence

time course most specific, sensitive and reproducible not much more expensive than conventional PCR

(except equipment cost)

The Basic of Real time PCR

Two method for quantify real time pcr results:

ABSOLUTE QUANTIFICATION&

RELATIVE QUANTIFICATION

Absolute quantification

Relative quantification This involves comparing the Ct values of the samples of interest with

a control or calibrator such as a non-treated sample or RNA from

normal tissue. The Ct values of both the calibrator and the samples

of interest are normalized to an appropriate endogenous

housekeeping gene(GAPDH ,rRNA ,…).

The comparative Ct method is also known as the 2-ΔΔCt method :

ΔΔct=Δct sample – Δct reference

Here, Δct,sample is the Ct value for any sample normalized to the

endogenous housekeeping gene and ΔCt, reference is the Ct value for

the calibrator also normalized to the endogenous housekeeping gene.

Detection in real time PCR Uses fluorescence as a reporter by Three

general methods :

1. DNA-binding agents

(SYBR Green)less accuracy.

2. Hydrolysis probes(TaqMan)

3. Hybridization probes

(Light Cycler) most accurate & specific.

Beacons, Scorpions

DNA binding dye Binds to minor groove

(dsDNA) Emits light when bound More double stranded DNA =

more binding = more fluorescence

Forensically, can be used to calculate how much DNA was present before reaction.

Unspecific Melting curve analysis

1.SYBR® green

The advantage of this technique is that it is

relatively cheap as it can be used with any pair

of primers for any target. However, as the

presence of any dsDNA generates

fluorescence, specificity of this assay is

greatily decrease due to amplification of

nonspescific PCR products and primer-dimers.

Generating and comparing melting curves

using the light cycler is one method of

increasing the specificity of the reaction.

Melting curve analysis

Hydrolysis probe(taqman)

Dual hybridization probe

Molecular beacon

scorpions

references

MT Rahman , MS Uddin , R Sultana , A Moue , M Setu .

Polymerase chean reaction (PCR) : A Short Rewiew . AKMMC J

2013: 4(1): 30-36

Manit A,Iqubal S ,Magali W ,Lyndon G et all .basic principles of

real-time quantification pcr .mol.diagn.5(2),209-219.(2005)

http://www.nature.com/nprot/journal/v1

http://www.cryst.bbk.ac.uk/pps97/assignments/projects

http://www.invitrogen.com