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Why is Real Time PCR Important

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Taq Extends from Upstream Primer. 5'-Nuclease Activity Digests Probe ... Shares primer set with target. Must use many ratios of mimic to target. ... – PowerPoint PPT presentation

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Title: Why is Real Time PCR Important


1
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.
2
SDS Methods
PCR amplification, detection and analysis
Assay design
Sample prep
No Gels!
3
Fluorescent Chemistry Choices
SYBR Green
TaqMan
4
TaqMan Uses the 5-Nuclease Assay and FRET
5
Taq Activities
5 nuclease
synthetic
6
5-Nuclease Activity Does Not Harm Normal PCR
1. Hydrogen-bonded 5-terminus. 2. Enzyme must
be associated with 3-OH.
7
TaqMan Uses FRET
Fluorescent Resonant Energy Transfer
8
TaqMan Uses a Fluorescent Probe
Reporter dye
Quencher dye
9
Taq Extends from Upstream Primer
10
5-Nuclease Activity Digests Probe
11
Increase in Reporter Signal Reports on
Amplification
Cycle number
12
Specificity Comparison
Gel
Blot
TaqMan
Band - size
Band - size Probe binds
Probe binds Primer upstream Primer same
strand Primer-probe distance Probe cleavage
13
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
14
Specificity
TaqMan
High specificity is inherent in TaqMan chemistry.
SYBR Green
Low specificity. Specificity check by melt
analysis.
15
ABI Prism 7700Sequence Detection System
CCD Camera
Laser Source
Heated Lid Thermal Cycler Assembly
16
Quantitative PCR Applications
Gene Expression Transgenics Cancer
quantitation DNA Damage Quality Control Pathogen
quantitation
17
PCR Phases
Plateau
Linear
Ethidium-Gel detection
Log DNA
Geometric
Cycle
18
Geometric Phase
y 2X
19
Problems with Linear and Plateau Phases
Variable Linear Phase
Plateau Effect
20
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.
.
Sample each cycle graph.
.
.
Limit cycle number.
.
.
21
What Real-Time Technology Does
Real-Time Techology provides easy access to high
quality geometric phase data.
Linear and plateau phases avoided.
22
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.
23
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.
24
Example of a Standard Curve
25
Thermal Cycling
26
Data is Imported Automatically
27
Amplification Plot
28
Standard Curve
y -3.6x 39 R2 0.999
7 pg DNA
700 ng DNA
29
Experiment Report
30
Fast Assay Development System
Design primers and probes using Primer Express.
Obtain PCR reagents, primers and probes.
TaqMan
Primer matrix.
SYBR
Assay ready.
31
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.
32
SDS Web Site
www.pebio.com/ab/about/pcr/sds
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