PPT Slide
Our New Address
Real-Time PCR detection
Basic principle of real-time PCR quantitation
A measure of when
Amplification plot features
Serial dilutions give serial amplification plots (setting the threshold is a crucial step!)
TaqMan probe
FRET Fˆster resonance energy transfer through space
Fluorogenic 5' nuclease assay (TaqMan_ chemistry)
ABI Prismô Fluorescent dyes
Changing fluorescence in multiplex PCR dual reporter raw data view
SYBR Green 1_
Normalising SYBR Green to the ROX passive reference ódistinguishing 5,000 and 10,000 copies
ABI Prism_ 7700 Sequence Detection System for real-time multicolour nucleic acid monitoring
ABI Prism_ 7700 Sequence Detection System optics of the virtual filter system
Sequence detection applications
Genotypingócompeting probes discrimination alleles and single nucleotide polymorphisms
Genotyping óapplication software
turbo TaqMan probes
Quantitation Options
Absolute quantitation example þ-actin install runóusing SYBR Green I dye on the 5700 instrument
þ-actin install run standards using SYBR Green I (linear view)
þ-actin install run standards using SYBR Green I (Log view)
þ-actin install run result
þ-actin install run result partial report for consecutive 12 replicates
Live Demonstration of SDS v 1.7
Relative Quantitation theory
_CT is a constant value
Converting _CT into a ratio
_CTs for 10 systems
Test for DNA contamination
DNA Contamination of kidney RT-PCR
Comparing relative results between treatments
96-well Tissue comparison experiment all samples multiplexed with the 18S rRNA target as the endogenous control
Expression levels relative to pancreas (here pancreas is the nominated __CT calibrator)
Relative Quantification Software Display: T cell Activation measured with the Cytokine Card
Easy Experimental Design!
Enzymology of real-time PCR
Live Primer Express Demonstration
Pre-Developed Assay Reagents (PDARs)
SDS as a flexible platform
End-point PCR with control eg. for detecting Salmonella
Dissociation curves Products from þ-actin Install run (raw view)
DNA dissociation curve detection of primer-dimer artifact
DNA dissociation curve detection of alleles
Author: Richard Harrison, PEBiosystems
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