Beruflich Dokumente
Kultur Dokumente
Quantitative PCR
Biotech Trait Detection Workshop
Ames, IA
May 8th-10th
Brian Coullahan
Field Applications Scientist
Brian.coullahan@stratagene.com
Presentation Outline
Review PCR fundamentals
Current systems used for GMO testing
Polymerase
Chain
Reaction
Components in a PCR
Template DNA
dNTPs
Forward and reverse primers
Thermal-stable DNA polymerase
Buffer (tris, KCl, Mg2+ , etc..)
Phases of PCR
[DNA]
Plateau phase
linear phase
Exponential phase
Cycle #
gDNA
gDNA
Gel-based Analysis
Sample
1
Insert-specific target
At what level?
Low range
of detection
Baseline
Am
pli
fic
ati
on
Fluorescence (R)
Threshold
Ct
Cycle #
Xn=X0 (1+E)n
Xn=X0 2n
X = DNA concentration
X0= Starting DNA concentration
n
Xn= DNA concentrationXat
= cycle
DNA concentration
E = Efficiency of PCR reaction
: 0Starting
E 1DNA concentration
X0=
Xn= DNA concentration at cycle n
Xn=X0 (1+E)n
X = DNA concentration
X0= Starting DNA concentration
Xn= DNA concentration at cycle n
E = Efficiency of PCR reaction, 0-1
[DNA]
Quantitative PCR
Threshold
15
Ct
Ct
Cycle #
Standard Curve
Efficiency= 99.5%
Fluorescence Detection
light
light
n
tio
p
r
so
b
A
iss
m
E
Wavelength
ion
Probe Based
Detection
TaqMan
Molecular Beacons
Lux primers
Hybridization probes
Scorpions
Amplifluor probes
FRET
TM
SYBR Green I
DNA + free dye
(weak fluorophore)
Activation
Amplification
Dissociation
Co
nt
ro
l- W
T
gD
N
Am
pl
ico
n
fro
m
in
se
rt
Co
nt
ro
l- W
T
gD
N
TaqMan Probes
"
"
"
$
%
%
&
% WT
% GMO
standard 1
99.04
0.96
standard 2
99.52
0.48
standard 3
99.76
0.24
standard 4
99.88
0.12
standard 5
99.94
0.06
standard 6
99.97
0.03
100
NTC
NTC
WT control
.48
0.12
0.03
.96 .24
0.06
% of total gDNA
Ct
Accurate quantitation of
% contamination
Log quantity
Standard Curve
Conclusion
PCR is a invaluable tool enabling the GMO environment
to reach levels of sensitivity unable to be obtained from
other methods
Quantitative PCR is the next technological step in
accurate detection and quantification of GMO testing in
food materials