Why is Real Time PCR Important

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Why is Real Time PCR Important?
Real time PCR addresses the needs of
high-throughput PCR and PCR applications
requiring precision.
Beta actin SYBR. Two groups of 36 replicates
each.
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SDS Methods
PCR amplification, detection and analysis
Assay design
Sample prep
No Gels!
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Fluorescent Chemistry Choices
SYBR Green
TaqMan
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TaqMan Uses the 5-Nuclease Assay and FRET
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Taq Activities
5 nuclease
synthetic
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5-Nuclease Activity Does Not Harm Normal PCR
1. Hydrogen-bonded 5-terminus. 2. Enzyme must
be associated with 3-OH.
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TaqMan Uses FRET
Fluorescent Resonant Energy Transfer
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TaqMan Uses a Fluorescent Probe
Reporter dye
Quencher dye
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Taq Extends from Upstream Primer
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5-Nuclease Activity Digests Probe
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Increase in Reporter Signal Reports on
Amplification
Cycle number
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Specificity Comparison
Gel
Blot
TaqMan
Band - size
Band - size Probe binds
Probe binds Primer upstream Primer same
strand Primer-probe distance Probe cleavage
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SYBR Green I
Believed to be a minor groove binding dye.
No probe lower reagent cost than TaqMan.
No more than 0.5?g of DNA in sample.
Cannot multiplex.
Minor groove binder Pico Green
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Specificity
TaqMan
High specificity is inherent in TaqMan chemistry.
SYBR Green
Low specificity. Specificity check by melt
analysis.
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ABI Prism 7700Sequence Detection System
CCD Camera
Laser Source
Heated Lid Thermal Cycler Assembly
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Quantitative PCR Applications
Gene Expression Transgenics Cancer
quantitation DNA Damage Quality Control Pathogen
quantitation
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PCR Phases
Plateau
Linear
Ethidium-Gel detection
Log DNA
Geometric
Cycle
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Geometric Phase
y 2X
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Problems with Linear and Plateau Phases
Variable Linear Phase
Plateau Effect
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Traditional Methods for Quantitative PCR
Use a competitor or mimic.
Shares primer set with target.
Competitor has altered size.
Must use many ratios of mimic to target.
Radioactive methods.
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Sample each cycle graph.
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Limit cycle number.
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What Real-Time Technology Does
Real-Time Techology provides easy access to high
quality geometric phase data.
Linear and plateau phases avoided.
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External Standard
Target nucleic acid amplified as a standard
curve to compensate for amplification efficiency
and provide quantity units.
target A
Detection threshold
target B
Log DNA
Cycle no.
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Advantages of External Standard
No site-directed mutagenesis.
No need to spike in competitor.
Better model of target.
No competition broad dynamic range.
One tube one result cost savings.
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Example of a Standard Curve
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Thermal Cycling
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Data is Imported Automatically
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Amplification Plot
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Standard Curve
y -3.6x 39 R2 0.999
7 pg DNA
700 ng DNA
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Experiment Report
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Fast Assay Development System
Design primers and probes using Primer Express.
Obtain PCR reagents, primers and probes.
TaqMan
Primer matrix.
SYBR
Assay ready.
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Benefits of the Fast Assay Development System
Defined assay development pathway.
Minimize assay development time effort.
All assays work under universal conditions.
Assays have robust performance.
Strong trouble-shooting support.
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SDS Web Site
www.pebio.com/ab/about/pcr/sds