What is Real-Time qPCR?

AMPLIFICATION

• Fluorescence-based detection of amplification products

through the use of a DNA-binding dye or hybridization
probe.

• Real-time qPCR is used to quantify input nucleic acid

by measuring the number of cycles required to reach a
set level of product.

• In contrast, traditional PCR is used to amplify DNA with

end point analysis to distinguish products.

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AMPLIFICATION
Limitations of standard PCR

Amplification is exponential, but the exponential increase

is limited:
• A linear increase follows Theoretical
exponential phase
•

Log Target DNA

Eventually plateaus Real Life
In theory, the amount of DNA
produced at every cycle should
double,
Product(T) = (Template0) x 2n
(n = # of cycles)

Cycle #
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AMPLIFICATION
Standard PCR is as endpoint

96 identical reactions will have very different final

amounts of fluorescence at endpoint

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AMPLIFICATION
Real-Time PCR

Through the use of fluorescent molecules,

real-time PCR has the ability to directly
measure the reaction while amplification is
taking place.

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AMPLIFICATION

How is quantitative data collected?

Real Life
Theoretical
Log Target DNA

Detector

Cycle #
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AMPLIFICATION
Threshold Cycle, CT

96 identical reactions will have

almost identical CT values

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AMPLIFICATION
Threshold Cycle, CT
The point at which the fluorescence rises
appreciably above background

Threshold can be placed anywhere in the exponential

(log-linear) phase

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 Threshold Setting
AMPLIFICATION

• After baseline subtraction, a threshold line is set empirically or by a

statistical calculation at a fluorescence value above background.

Threshold

Log
View

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 Mathematical Implications
AMPLIFICATION

Ideal PCR
n
ProductT=(Template0)2
Where n=Number of Cycles

• 1 CT Difference = 2 fold difference in starting template amount

• 3.3 CT Difference = 10 fold difference in starting template amount

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AMPLIFICATION
Threshold Cycle, CT
• Correlates strongly with the starting copy number

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AMPLIFICATION
Threshold Cycle, CT
• Correlates strongly with the starting copy number

2n = 10 fold
n ln 2 = ln 10

n = ln10
ln 2

n = 3.32

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 Real-Time PCR: Applications
AMPLIFICATION

Real-Time reaction monitoring provides information for

relative or absolute
measurements of starting material.

• Gene Expression Studies

• Chromatin Immunoprecipitation (ChIP)
• Methylation Specific PCR (HRM)
• Microarray Validation
• Transgenic Analysis
• GMO Testing
• Viral/Bacterial Load Studies
• Allelic Discrimination/SNP (HRM)

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 From CT values, we can determine
AMPLIFICATION the initial copy number

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AMPLIFICATION

Chemistries used in real time PCR

• Intercalation Dyes
• Hybridization Probes

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AMPLIFICATION

Intercalation (DNA binding) dyes

 DNA binding dyes are inexpensive compared to
hybridization probes.

 EtBr is 25 times more fluorescent when bound to

dsDNA

 SYBR Green I is 125 times more fluorescent brightly

bound to dsDNA

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AMPLIFICATION
Intercalation Dyes:
SYBR Green I

l l l

3’
Taq 5’
ID ID ID

ID
ID 3’
Taq

l
l

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 SYBR Green I
AMPLIFICATION

• Advantages
– Experiment only requires primers

• Disadvantages
– Potential contribution to fluorescence from non-
specific products (primer-dimers)
– No multiplexing

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AMPLIFICATION
Hybridization Probes

Currently, hybridization probe strategies

fall into three main categories:
• Cleavage-based assay
• TaqMan Assays
• Locked nucleic acids (LNA)
• Displaceable probe assays
• molecular beacons
• Dual-oligo FRET probes
• Probes incorporated directly into the primers
• Amplifluor & Scorpions

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 Cleavage-based assay: TaqManTM
AMPLIFICATION

3’ 5’
5’ 3’

Add iQ Supermix, d.NTPs

Primers
Hybridization Probe Thermal Stable
DNA Polymerase
and sample R Q
5’ Probe 3’

Denaturation

Taq

l Q
R
5’ 3’

Annealing
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AMPLIFICATION
Cleavage-based assay: TaqManTM
Q R

5’ 3’

R
Q
Taq
Extension Step 5’ 3’
R

Q
Taq
5’
5’ 3’

R
Taq
5’
5’ 3’

l
R
Taq
5’
5’ 3’

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 TaqMan
AMPLIFICATION

• Advantages
– Target specific fluorescence
– Multiplexing

• Disadvantages
– High initial cost
– Assay design not trivial

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 Melt Curve Analysis
AMPLIFICATION

• After real-time PCR amplification, a melt curve is performed in presence

of a DNA binding “saturation dye”

• Melting temperature (Tm)

– DNA is half double and half single-stranded
– Depends on nucleotide content and length

Double Single
Stranded DNA Stranded

Tm

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 Melt Curve Analysis
AMPLIFICATION

Endpoint analysis to determine the melting

temperature (Tm) of PCR products.

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 Melt Curve Analysis:
AMPLIFICATION
Primer Dimer

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