Real Time PCR

Real-Time PCR
1.Why Real-time PCR? Advantages and Disadvantages 2.Theory of Real-time PCR 3.Types of Real-time PCR Quantification 4.Choosing Housekeeping Gene for Normalization

1. Why Real-time PCR ?

Disadvantage of traditional PCR

* * * * * * * Low sensitivity Short dynamic range Low resolution Non-automated Size-based discrimination only Results are not expressed as numbers Ethidium bromide staining is not very quantitative

1. Why Real-time PCR ?

Advantages of real-time PCR

amplification can be monitored real-time wider dynamic range of up to 1010-fold no post-PCR processing of products (No gel-based analysis at the end of the PCR reaction) ultra-rapid cycling (30 minutes to 2 hours) highly sequence-specific

1. Why Real-time PCR ?

Disadvantages of real-time PCR

1.Requires reagents expensive equipments and

2.Due to its extremely high sensitivity, you may get high deviations of the same experiment, thus, the use of internal control genes is a recommended (in gene expression experiments)

2. Theory of real-time PCR

2- Theory of Real-time PCR ?

Q-PCR
Definition: Real-time monitoring of the amplification reaction. Purpose: To estimate the initial quantity of specific template DNA.

2- Theory of Real-time PCR ?

The QPCR Approach

Chemistry
l l

Use fluorescent dyes and probes Establish a linear correlation between PCR product and fluorescence intensity Fluorescence detection to monitor amplification in real time Software for analysis and estimation of template concentration

Detection
l

Analysis
l

Reaction contents
10 X NH4 Buffer dNTP mix (12.5 mM) each Forward primer (20 M) Reverse primer (20 M) MgCl2 Sterile Milli-Q water Taq polymerase 5.0 l 0.8 l 1.0 l

1.0 l

3.0 l 38.0 l 0.5 l

10 X NH4 Buffer 5.0 l dNTP mix (12.5 mM) each 0.8 l Forward primer (20 M) 1.0 l Reverse primer (20 M) 1.0 l MgCl2 3.0 l Sterile Milli-Q water 37.0 l Taq polymerase 0.5 l SybrGreen (50x) 1.0 l

2- Theory of Real-time PCR ?

Chemistry

2- Theory of Real-time PCR ?

How to measure the PCR product

Directly
Sybr green Quality of primers critical

Indirectly
In addition to primers, add a fluorescently labeled hybridization probe

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2- Theory of Real-time PCR ?

Chemistries
TaqMan Molecular Beacons ScorpionsTM Amplifluor probes SYBR Green ITM Others...

2- Theory of Real-time PCR ?

Chemistry

Detection Chemistry

Primer & Probe

Primer
Short (Often < 50 nt) oligonucleotide sequence of DNA Complementary to the beginning and the end of the target DNA sequence Needed to initiate the synthesis of new DNA in a PCR reaction Involved in AMPLIFICATION

Probe Primer

Probe
A single-stranded DNA with a specific base sequence Labeled with fluorescence dyes (TaqMan probe) Used to detect the complementary base sequence of target DNA/RNA by hybridization Involved in DETECTION Reporter dye / Quencher dye

Detection Chemistry

DNA binding agents - Intercalating method: SYBRGreen I

Fluorescent dyes
Hydrolysis Probe - TaqMan probe, Molecular Beacon Hybridization Probe - Dual oligo FRET probes Primer based Probe - Scorpion

SYBR Green dye

Intercalating method 1) Denaturation
F F F F

1)

Intercalating dye fluoresces more brightly when bound to dsDNA.

2) Annealing
F F F F F

2)

DNA binding dyes are inexpensive compared to the other probes.

3) Extension
3)
F F F

SYBR Green I, EtBr

SYBR Green dye

Intercalating method 1. Advantages:
- Cheap, easy to use - Does not inhibit the reaction of amplification - Does not require any fluorescent probe - Does not require any particular expertise for the design of the probes - Is not affected by mutations in the target DNA

2. Disadvantages:
- Impossible to make sure of specificity of amplicons - Bad pairing can lead to positive forgeries or an over-estimate of the quantification

- Still unspecified mutagen capacity

TaqMan Probe
TaqMan probe 1) Denaturation
1)
F Q

Fluorescent reporter dye at the 5 end is quenched by fluorescent quencher dye at the 3 end.

2) Annealing
Taq F Q

2)

3) Extension
F Q

When amplification occurs the TaqMan probe is degraded due to the 5'-->3' exonuclease activity of Taq DNA polymerase, thereby separating the quencher from the reporter during extension.

3)

The TaqMan assay accumulates a fluorescence signal.

TaqMan Probe
TaqMan probe 1. Advantages:
- Increased specificity - Better capacity of multiplexing

2. Disadvantages:
- Little expensive (dual-labeled probe) - Less effective and less flexible compared to other techniques in the real-time detection of specific mutation - Require the design of probes

Molecular Beacon Probe

Molecular Beacon 1) Denaturation
F Q

1)

2) Annealing
Taq

A molecular beacon begins as a stem-and-loop structure. The sequences at the ends of the probe match and bind, creating the stem

2)

3) Extension
F Q

When the probe binds to a singlestranded DNA template, the structure unfolds, separating the quencher from the dye and allowing fluorescence.

Molecular Beacon Probe

Molecular Beacon 1. Advantages:

- Increased specificity - High flexibility for probe design - As the probes are not hydrolyzed, they are used at each cycle

2. Disadvantages:
- Little expensive (dual-labeled probe) - Less effective and less flexible compared to other techniques in the real-time detection of specific mutation - Require the design of probes

FRET Probe
Hybridization Probe (FRET) 1) Denaturation
F F

1)

FRET method designed two specifically probe. It labeled with different dyes, such as at the 5 end of donor probe and at the 3 end of acceptor probe.

2) Annealing
Taq F F

Energy transfer

2)

3) Extension
F F

At close proximity, the donor dye is excited by the light source and the energy is transferred the acceptor dye. Subsequently, fluorescent light is emitted at a different wavelength.

2- Theory of Real-time PCR ?

Fluorescence Detection
Light Light

Excitation

Emission

2- Theory of Real-time PCR ?

REAL TIME PCR

USING SYBR GREEN

2- Theory of Real-time PCR ?

Sybr green is a dye which binds to double stranded DNA but not to single-stranded DNA and is frequently used to monitor the synthesis of DNA during real-time PCR reactions. When it is bound to double stranded DNA it fluoresces very brightly (much more brightly than ethidium bromide does

2- Theory of Real-time PCR ?

SYBR Green Assay

SYBR Green SYBR Green SYBR Green (high fluorescent conformation) SYBR Green SYBR Green SYBR Green SYBR Green

2- Theory of Real-time PCR ?

Q RT PCR Using TaqMan

The TaqMan probe principle relies on the 53 nuclease activity of Taq polymerase to cleave a dual-labelled probe during hybridization to the complementary target sequence and fluorophore-based detection. TaqMan probes consist of a fluorophore (Reporter) attached to the 5-end of the oligonucleotide probe and a quencher at the 3-end

2- Theory of Real-time PCR ?

The quencher molecule quenches the fluorescence emitted by the reporter when excited by the cyclers light source via FRET (Fluorescence Resonance Energy Transfer). As long as the reporter and the quencher are in proximity, quenching inhibits any fluorescence signals

2- Theory of Real-time PCR ?

TaqMan Chemistry

2- Theory of Real-time PCR ?

TaqMan Chemistry
R R R R R R R

2- Theory of Real-time PCR ?

TaqMan Chemistry
R

2- Theory of Real-time PCR ?

TaqMan Chemistry
R

2- Theory of Real-time PCR ?

Molecular Beacons

R R R

QQ Q Q Q

2- Theory of Real-time PCR ?

SCORPIONS

2- Theory of Real-time PCR ?

SCORPIONS
Q Q Q R R Q RR R

R
R R

RQ Q Q Q

2- Theory of Real-time PCR ?

Comparison of Probe Chemistries

Taqman

Beacon

Scorpion

2- Theory of Real-time PCR ?

Published References for real-time PCR Fluorescent Chemistries
180 160 140 120

No. of references

100 80 60 40 20 0 1994 1995 1996 1997 Year 1998 1999 2000 2001

Taqman SYBR Green molecular beacons scorpions

2- Theory of Real-time PCR ?

Optical Detection System of RealTime PCR

1. halogen tungsten lamp

3. intensifier

2b. emission filters 2a. excitation filters

5. detector 350,000 pixels

4. sample plate

www.biorad.com

2- Theory of Real-time PCR ?

Quantitative PCR
[DNA]

Threshold

Limit of detection

Ct Cycle #

Linear ground phase: 2. Theory of Real-time PCR PCR is just began Fluorescence emission at each cycle has not yet risen above background Baseline fluorescence is calculated at this time CT - threshold cycle: the first significant increase in the amount of PCR product correlates to the initial amount of target template CT represents the starting copy no. in the original PCR cantemplateinto 4 major phases be broken

Early exponential phase: PCR is just began The amount of fluorescence has reached a threshold where it is significantly higher than background (usually 10 times the standard deviation of the baseline)

2- Theory of Real-time PCR ?

Advantage of Real-Time PCR

Real-time PCR vs. Conventional PCR

Real-Time PCR
Sensitivity Specificity Quantitative results Detection method Detection range Reaction time Post-PCR steps Crosscontamination High High -use specific probes Yes -Specific fluorescence Probe-specific Fluorescence Wide range 1 hr No No -Closed system - Single step

PCR
Low Low -only size discrimination No -EtBr staining Agarose gel Electrophoresis Short range (<2 log) 3-5 hr Agarose gel electrophoresis Yes -Open system - Multiple steps

Glossary of Real-Time PCR

Amplification plot: The plot of cycle number vs. fluorescence signal which
correlates with the initial amount of target nucleic acid during the

exponential phase of PCR

Baseline: The initial cycles of PCR during which there is little change in
fluorescence signal (usually cycles 3 to 15)

Amplification plot

Fluorescence

Ct

Threshold

Baseline

cycle

Glossary of Real-Time PCR

Threshold: the fluorescence measurement at which product can be
distinguished from background. Threshold should be set in the region associated with an exponential growth of PCR product

Ct (Threshold cycle): The cycle number at which the fluorescence generated

within a reaction crosses the threshold. It is inversely correlated to the log of he initial copy number

Amplification plot

Fluorescence

Ct

Threshold

Baseline cycle

Types of Real Time PCR Quantification

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3. Types of Real-time PCR Quantification

STANDARD CURVE METHOD

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1600000000 1600000000
AMOUNT OF DNA

1200000000 1200000000 1000000000 1000000000 800000000 600000000 400000000 200000000 0 0 5 800000000 600000000 400000000 200000000 0 0 10 5 15 10 20 15 25 20 30 25 35 30 35 PCR CYCLE NUMBER PCR CYCLE NUMBER

AMOUNT OF DNA

1400000000 3. Types of Real-time PCR Quantification

1400000000

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SERIES OF 10-FOLD DILUTIONS

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threshold

Ct

3. Types of Real-time PCR Quantification

SERIES OF 10-FOLD DILUTIONS

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3. Types of Real-time PCR Quantification

copy number reference gene experimental copy number reference gene control

Dilution curve reference gene

NORTHERN
control expt

target gene internal control gene actin, GAPDH, RPLP0 etc

Ratio experimental/control = fold change in target gene fold change in reference gene
4

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DDCt EFFICIENCY METHOD

APPROXIMATION METHOD

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IL1-b vit
RPLP0 con RPLP0 vit IL1-b con

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control

RPLP0 con

D Ct = target - ref
D Ct = 9.70

IL1-b con
av =19.93 av =29.63

experiment

IL1-b vit RPLP0 vit

D Ct = target - ref D Ct = -1.7 Difference = DCt-DCt = DDCt = 9.70-(-1.7) = 11.40

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av =18.03 av =19.80

4- Choosing Housekeeping Gene for Normalization

Standards
same copy number in all cells expressed in all cells no pseudogene no alternate splicing in target PCR region you want to amplify.

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4- Choosing Housekeeping Gene for Normalization

Standards

Commonly used standards

Glyceraldehyde-3-phosphate dehydrogenase mRNA Beta-actin mRNA MHC I (major histocompatability complex I) mRNA Cyclophilin mRNA mRNAs for certain ribosomal proteins

E.g. RPLP0 (ribosomal protein, large, P0; also known as 36B4, P0, L10E, RPPO, PRLP0, 60S acidic ribosomal protein P0, ribosomal protein L10, Arbp or acidic ribosomal phosphoprotein P0)
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28S or 18S rRNA

4- Choosing Housekeeping Gene for Normalization

Standards

The perfect standard does not exist

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Applications of Q RT PCR

Gene expression (and microarray validation). DNA target quantification (nuclear, mitochondrial, residual DNA in protein preps (QC)). SNP detection, Allele discrimination, Genotyping, Haplotyping DNA Methylation, Apoptosis Viral load assays, pathogen & GMO detection. Clinical Diagnostics (Cancer, Therapy Response)