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An Introduction to PCR and An Introduction to PCR and Quantitative PCRQuantitative PCR

Brian CoullahanField Applications Scientist

[email protected]. Services 800-894-1304

Biotech Trait Detection WorkshopAmes, IA

May 8th-10th

Presentation Outline

Review PCR fundamentalsCurrent systems used for GMO testing

Introduction to Quantitative PCRQuantitative 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 moleculePCR has allowed for simplification of techniques such as cloning, target detection, sequencing, etc

Polymerase Chain

Reaction

Template DNAdNTPsForward and reverse primersThermal-stable DNA polymeraseBuffer (tris, KCl, Mg2+ , etc..)

Components in a PCR

Phases of PCR[

D

N

A

]

Cycle #

Exponential phase

linear phase

Plateau phase

insertioninsertion

gDNAgDNA

Detection of Insertion using PCR

Foreign DNA is inserted into genome of organism

gDNAgDNA

Detection of Insertion using PCR

30+ cycles to reach plateau phase

Insert-specific target

Control from WT gDNA

Sample1 2 3 4 5 6

Gel-based Analysis

Low range of detection

Insert was detected in sample1 and sample3At what level?

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.

Cycle #

F

l

u

o

r

e

s

c

e

n

c

e

(

R

)

Baseline

CCtt = Fractional PCR cycle number at which the fluorescence intensity crosses the established threshold line.

Ct

Threshold

QPCR Amplification Plot

Ampli

ficatio

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)nX = DNA concentration

X0= Starting DNA concentrationXn= DNA concentration at cycle n

E = Efficiency of PCR reaction : 0 E 1

Xn=X0 2nX = DNA concentration


PCR Molecular Mechanism Exponential amplification of the original DNA

sequence (template) to create copies of part of the sequence (amplicon)

Xn=X0 (1+E)nX = DNA concentration


E = Efficiency of PCR reaction, 0-1

Quantitative PCR[

D

N

A

]

Cycle #

Threshold

15

Ct

Ct

Standard Curve

Efficiency= 99.5%

Absorp

tion

light

Emissi

on

light

Fluorescence DetectionFluorescence Detection

Wavelength

Quantitative PCR Chemistries SYBR Green ITM

TaqMan Molecular Beacons Lux primers Hybridization probes ScorpionsTM Amplifluor probes FRET

dsDNA Binding

Probe Based Detection

DNA + free dyeDNA + free dye(weak (weak fluorophorefluorophore))

Binds minor groove Binds minor groove dsDNAdsDNA(fluorescence (fluorescence 1,000x)1,000x)

SYBR Green I

SYBR Green I Thermal ProfileSYBR Green I Thermal Profile

Activation Amplification Dissociation

SYBR Green I DetectionSYBR Green I Detection

Cont

rol-

WT

gDNA

End-Point Melt Curve Detection

Cont

rol-

WT

gDNA

Ampli

con

from

inse

rt

End-Point Melt Curve Detection

TaqMan Probes

!

"

"#$

%%&

"

!

!

44--Fluor MultiplexFluor Multiplex--Standard CurvesStandard Curves

44--Fluor MultiplexFluor Multiplex--Standard CurvesStandard Curves

%E Cy5=95.3%%E Rox=98.9%%E Hex=101.3%%E Fam=91.7%

0100NTC0.0399.97standard 60.0699.94standard 50.1299.88standard 40.2499.76standard 30.4899.52standard 20.9699.04standard 1

% GMO% WTDilution series of GMO product in required dynamic range

Standards diluted in WT gDNAmatrix

QPCR for GMO Detection

0.060.12

.24.48

.96

% of total gDNA

WT control0.03


NTC

WT specific amplicon

GMO specific amplicon

Ct

Log quantity

Accurate quantitation of % contamination

< lowest dilution in curve

> Highest dilution in curve


Standard Curve Analysis

Standard Curve

Results from Standard Curve

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

Conclusion

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