An Introduction to PCR and Quantitative PCR

An Introduction to PCR and
Quantitative PCR
Biotech Trait Detection Workshop
Ames, IA
May 8 th -10 th
Brian Coullahan
Field Applications Scientist
Brian.coullahan@stratagene.com
Tech. Services 800-894-1304

Presentation Outline
• Review PCR fundamentals
Current systems used for GMO testing
• Introduction to Quantitative PCR
Quantitative methods for GMO testing

Polymerase Chain Reaction
Introduced to the Scientific community in 1983,
PCR allows for exponential amplification of
sequence-specific targets in a DNA molecule
PCR has allowed for simplification of techniques
such as cloning, target detection, sequencing,
etc…

Polymerase
Chain
Reaction

Polymerase
Chain
Reaction

Components in a PCR
•Template DNA
•dNTPs
•Forward and reverse primers
•Thermal-stable DNA polymerase
•Buffer (tris, KCl, Mg 2+ , etc..)

Phases of PCR
Plateau phase
[DNA]
linear phase
Exponential phase
Cycle #

Detection of Insertion using PCR
insertion
gDNA
Foreign DNA is inserted into genome of organism

Detection of Insertion using PCR
gDNA
30+ cycles to reach plateau phase

Gel-based Analysis
Sample
1 2 3 4 5 6
Insert-specific target
Control from WT gDNA
Insert was detected in
sample1 and sample3
At what level
Low range
of detection

Quantitative Real Time PCR
• Definition: Assay that monitors the
accumulation of a DNA product from a
PCR reaction.
– Quantitate the initial number of copies of a
particular DNA in a sample.
– Benefits from improved sensitivity,
reproducibility, dynamic range, throughput,
cost.

QPCR Amplification Plot
Fluorescence (R)
Baseline
Amplification
Threshold
Ct
Cycle #
C t = Fractional PCR cycle number at which
the fluorescence intensity crosses the
established threshold line.

PCR Molecular Mechanism
• Exponential amplification of the original DNA
sequence (template) to create copies of part of the
sequence (amplicon)
X n =X 0 (1+E) n
X n =X 0 2 n
X = DNA concentration
X 0 = Starting DNA concentration
X n = DNA concentration X at = cycle DNA concentration
n
E = Efficiency of PCR reaction X 0 = : 0 Starting ≤ E ≥ 1DNA concentration
X n = DNA concentration at cycle n

PCR Molecular Mechanism
• Exponential amplification of the original DNA
sequence (template) to create copies of part of the
sequence (amplicon)
X n =X 0 (1+E) n
X = DNA concentration
X 0 = Starting DNA concentration
X n = DNA concentration at cycle n
E = Efficiency of PCR reaction, 0-1

Quantitative PCR
[DNA]
Threshold
Ct
Ct
15
Cycle #

Standard Curve
Efficiency= 99.5%

Fluorescence Detection
light
light
Absorption
Emission
Wavelength

Quantitative PCR Chemistries
dsDNA Binding
Probe Based
Detection
• SYBR Green I TM
• TaqMan ®
• Molecular Beacons
• Lux ® primers
• Hybridization probes
• Scorpions TM
• Amplifluor ® probes
• FRET

SYBR Green I
DNA + free dye
(weak fluorophore)
Binds minor groove dsDNA
(fluorescence ↑1,000x)

SYBR Green I Thermal Profile
Activation Amplification Dissociation

SYBR Green I Detection

End-Point Melt Curve Detection
Control- WT gDNA

End-Point Melt Curve Detection
Control- WT gDNA
Amplicon from insert

TaqMan ® Probes
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4-Fluor Multiplex-Standard Curves

4-Fluor Multiplex-Standard Curves
%E Cy5=95.3%
%E Rox=98.9%
%E Hex=101.3%
%E Fam=91.7%

QPCR for GMO Detection
•Dilution series of GMO product in
required dynamic range
standard 1
standard 2
% WT
99.04
99.52
% GMO
0.96
0.48
standard 3
99.76
0.24
•Standards diluted in WT gDNA
matrix
standard 4
standard 5
standard 6
99.88
99.94
99.97
0.12
0.06
0.03
NTC
100
0

QPCR for GMO Detection
WT specific amplicon
GMO specific amplicon
WT control
.96
.48
.24
0.12 0.03
0.06
NTC
% of total gDNA

QPCR for GMO Detection
< lowest dilution in curve
Ct
Accurate quantitation of
% contamination
> Highest dilution in curve
Log quantity

Standard Curve Analysis

Standard Curve

Results from 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