Title: Why is Real Time PCR Important
1Why 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.
2SDS Methods
PCR amplification, detection and analysis
Assay design
Sample prep
No Gels!
3Fluorescent Chemistry Choices
SYBR Green
TaqMan
4TaqMan Uses the 5-Nuclease Assay and FRET
5Taq Activities
5 nuclease
synthetic
65-Nuclease Activity Does Not Harm Normal PCR
1. Hydrogen-bonded 5-terminus. 2. Enzyme must
be associated with 3-OH.
7TaqMan Uses FRET
Fluorescent Resonant Energy Transfer
8TaqMan Uses a Fluorescent Probe
Reporter dye
Quencher dye
9Taq Extends from Upstream Primer
105-Nuclease Activity Digests Probe
11Increase in Reporter Signal Reports on
Amplification
Cycle number
12Specificity Comparison
Gel
Blot
TaqMan
Band - size
Band - size Probe binds
Probe binds Primer upstream Primer same
strand Primer-probe distance Probe cleavage
13SYBR 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
14Specificity
TaqMan
High specificity is inherent in TaqMan chemistry.
SYBR Green
Low specificity. Specificity check by melt
analysis.
15ABI Prism 7700Sequence Detection System
CCD Camera
Laser Source
Heated Lid Thermal Cycler Assembly
16Quantitative PCR Applications
Gene Expression Transgenics Cancer
quantitation DNA Damage Quality Control Pathogen
quantitation
17PCR Phases
Plateau
Linear
Ethidium-Gel detection
Log DNA
Geometric
Cycle
18Geometric Phase
y 2X
19Problems with Linear and Plateau Phases
Variable Linear Phase
Plateau Effect
20Traditional 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.
.
Sample each cycle graph.
.
.
Limit cycle number.
.
.
21What Real-Time Technology Does
Real-Time Techology provides easy access to high
quality geometric phase data.
Linear and plateau phases avoided.
22External 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.
23Advantages 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.
24Example of a Standard Curve
25Thermal Cycling
26Data is Imported Automatically
27Amplification Plot
28Standard Curve
y -3.6x 39 R2 0.999
7 pg DNA
700 ng DNA
29Experiment Report
30Fast Assay Development System
Design primers and probes using Primer Express.
Obtain PCR reagents, primers and probes.
TaqMan
Primer matrix.
SYBR
Assay ready.
31Benefits 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.
32SDS Web Site
www.pebio.com/ab/about/pcr/sds