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IntroPCR Brian

This document provides an overview of polymerase chain reaction (PCR) and quantitative PCR (qPCR) techniques for genetically modified organism (GMO) testing. It discusses how PCR allows exponential amplification of DNA sequences, and how qPCR monitors amplification in real-time to quantify initial DNA copies. qPCR uses fluorescent dyes or probes to detect amplification, and standard curves relate quantification cycle values to initial template amounts. The document demonstrates how qPCR can accurately quantify GMO content by analyzing dilution series against wild-type controls.

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71 views

IntroPCR Brian

This document provides an overview of polymerase chain reaction (PCR) and quantitative PCR (qPCR) techniques for genetically modified organism (GMO) testing. It discusses how PCR allows exponential amplification of DNA sequences, and how qPCR monitors amplification in real-time to quantify initial DNA copies. qPCR uses fluorescent dyes or probes to detect amplification, and standard curves relate quantification cycle values to initial template amounts. The document demonstrates how qPCR can accurately quantify GMO content by analyzing dilution series against wild-type controls.

Uploaded by

333
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© © All Rights Reserved
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Download as PDF, TXT or read online on Scribd
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An Introduction to PCR and

Quantitative PCR
Biotech Trait Detection Workshop
Ames, IA
May 8th-10th

Brian Coullahan
Field Applications Scientist

[email protected]

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

       



    


 
 
     
      

   

      
 

      
 

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 #

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

Insert was detected in


sample1 and sample3

Insert-specific target

At what level?

Control from WT gDNA

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.

Baseline

Am
pli
fic
ati
on

Fluorescence (R)

QPCR Amplification Plot

Threshold
Ct

Cycle #

Ct = 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)

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

PCR Molecular Mechanism


Exponential amplification of the original DNA
sequence (template) to create copies of part of the
sequence (amplicon)

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

Quantitative PCR Chemistries


dsDNA Binding

SYBR Green ITM

Probe Based
Detection

TaqMan
Molecular Beacons
Lux primers
Hybridization probes
Scorpions
Amplifluor probes
FRET
TM

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

Co
nt
ro
l- W
T

gD
N

End-Point Melt Curve Detection

Am

pl
ico
n

fro
m

in
se
rt

Co
nt
ro
l- W
T

gD
N

End-Point Melt Curve Detection

TaqMan Probes

"

"

"

$
%
%
&

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
Standards diluted in WT gDNA
matrix

% 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

QPCR for GMO Detection


WT specific amplicon
GMO specific amplicon

NTC
WT control

.48
0.12
0.03
.96 .24
0.06
% 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

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