Qpcr

QPCR Applications using Stratagene’s
QPCR
Mx Real-Time PCR Platform

Dan Schoeffner, Ph.D
Field Applications Scientist
Dan.Schoeffner@Stratagene.com
Tech. Services 800-894-1304

Polymerase
Polymerase
Chain
Reaction

DNA
Melt
Anneal primers

Extension/Measure

Melt
Anneal

+

Gene of
interest
(Amplicon)
Forward and
Reverse Primers

Q PCR Molecular MechanismExponential amplification of the original DNA sequence (template)
to create copies of part of the sequence (amplicon)

Xn=X0 (1+E)n
2n
X = DNA concentration
X0= Starting DNA concentration
Xn= DNA concentration at cycle n
E = Efficiency of PCR reaction, 0-1

http://allserv.rug.ac.be/~avierstr/principles/pcr.html

Influence of Reaction Efficiency
Influence

lifi
ca
tio
n

BaselineAm
p

Fluorescence (R)

Typical PCR Amplification Plot

Raw Signal (R)

Threshold
Ct

Cycle #

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

• A 1Ct difference between samples represents 2x more transcript
2x

Why is QPCR superior to PCR
Why
96 technical replicates

Variability using
QPCR

Variabilityusing
endpoint PCR

Gel-based quantification
Gel
1
2
1

2

Real-time quantification
Real
1
2

Unpredicable Amplification Plots with
Unpredicable
Endpoint Analysis

Chemistries used in QPCR
Chemistries
TET
TET
Alx350
Alx350

350nm

FAM
FAM

HEX TAM
HEX TAM
JOE Cy3
JOE Cy3

TxRd
TxRd

ROX
ROX

Cy5
Cy5

700nm

Fluorescence Detection
Light
λ

Lightλ
n
io
pt
r
so
Ab

n
s io
is
m
E

Quantitative PCR Chemistries
Quantitative
dsDNA Binding
Probe Based
Detection

SYBR Green
TaqMan®
Molecular Beacons
Lux® primers
Hybridization probes
ScorpionsTM
Amplifluor® probes
FRET

Chemistries
Chemistries
SYBR green
dsDNA binding dyes

Pro:
■ Ease of use
■ Inexpensive
■ Good for high throughput screenings
lots ofgenes: this is your chemistry
■ Great for first screens and optimization
■ Can detect amplicon heterogenity

Con:

1000x increase in
fluorescence

■ Sequence unspecific – detects any
double strand in your reaction
■ Can not multiplex reactions

Primer Selection
Primer
•

Try to achieve similar Tm for all primers: Ideal ~60°C.
(Future multiplexing or use of Taqman™ assays in mind)•

Forward and reverse primer should have ∆Tm -4
kcal/mol to avoid stable primer dimers

•

Design via software (Always use the same one):

•

Always perform a BLAST search with your amplicon and
primers
( Specificity of the PCR)

SYBR green
SYBR
Raw Fluorescence [R]

SYBR Green I™ Thermal Profile

Negative First Derivative [-R’(T)]

Activation AmplificationDissociation

Chemistries
Chemistries
Taqman probes
Pro:

R

Taq

■ Sequence specific
■ Possibility to do multiplex
- have GOI and normalizer in the same
well, doing comparative quantification

Q

R

Q

Con:
■ More difficult to design
■ Expensive

R
Q
Taq

Linear Taqman Probe Design
Linear
•
•
•
•
•
•

Probe Tm 5-10°C higher than primers
≤ 30 bp in length
No G nextto reporter fluorophore
< 4 contiguous Gs
PCR blocker at 3’ end
Compatible reporters and quenchers

Linear Taqman Probe Modifications
Linear
Increase thermal duplex stability

DNA

LNA

Improve specificity
Raise Tm by up to 8°C per LNA

O

Allow shorter probe design (~13bp)
(www.proligo.com)

Base

O

O

O

O
O

P

Base

O

O-

O

O

P

O-

2'-O,4'-C methylene bridge locks
conformation

Introduction to MxPro software
Introduction
Critical Setting
•Threshold
•Baseline

Analysis term settings
Analysis
Algorithms
• Developed using real Q-PCR training data, establish
settings and ranges
• Performs optimally for the majority of the
fluorescence signal analyzed
• Allows a user to analyze the raw data using the same
method...