Download Chemically Modified Primers for Improved Multiplex PCR

Chemically Modified Primers for Improved Multiplexed PCR
Elena Hidalgo Ashrafi, Tony Le, Alexandre Lebedev, Richard Hogrefe, Victor Timoshchuk, Sabrina Shore, Inna Koukhareva and Natasha Paul
Abstract
Multiplex PCR is an advantageous technique used in PCR
Figure 3
Figure 6
Figure 9
Real-time analysis of multiplex PCR using CleanAmp™
Turbo Primers.
Increasing multiplex amplification using CleanAmp™
Turbo Primers
Compatibility of CleanAmp™ Precision
Primers with different RT priming approaches
applications to amplify multiple targets in a single reaction. As useful as it is,
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setup. For example, reactions are more prone to off-target amplifications such
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533 bp
35
Cycles
this technique presents a new set of challenges that further complicates PCR
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25
25
20
20
15
1.E+01
as mis-priming and primer dimer due to the increased number of primer pairs.
1.E+02
1.E+03
1.E+04
1.E+05
600 bp
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30
Cycles
1.E+03
1.E+04
30
1.E+05
and yield in the multiplex amplification of DNA targets. In reverse
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x
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x
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1.E+06
1.E+02
1.E+03
1.E+04
1.E+05
for multiple targets. TriLink’s innovative technology represents a convenient
tool for multiplex PCR amplification of DNA and RNA samples.
Turbo Primers provide increased sensitvity in Real-time PCR.
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8
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Turbo Primers can efficiently amplify at least nine targets
ranging from 139 to 962 bp.
Thermal cycling conditions:
95oC (10 min); [95oC (40 sec), 56oC (30 sec), 72oC (2 min)]40X
Thermal cycling conditions:
95oC (10 min); [95oC (40 sec), 56oC (30 sec), 72oC (2 min)]35X, 72oC (7 min)
Turbo**
0.5
1
a
primer
concentration
(PM)
(
)
primer
concentration
(PM)
0.1
0.5
1
primer
concentration
(PM)
(
)
1
c
0.1
e
f
0.5
g
h
i
1
a
b
c
0.1
d
e
f
0.5
g
h
i
0.1
962bp. amplicon
962bp. amplicon
0.5
b
d
600bp. amplicon
0.1
0.1
0.5
1
0.5
1
5’
3’
1
1
0.5
X
O
a b c
dNTPs
5’
3’
O
“Hot Start”
Activation
O
O
O
Target
LEGEND
B2
Success
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x
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x
O
B1
PCR conditions:
1X PCR buffer (20 mM Tris (pH 8.4), 50 mM KCl, 2.5 mM MgCl2), Primers (0.5 μM), 0.2
mM dNTPs, 50,000 copies Lambda gDNA, 1.25 U Taq DNA polymerase, 50 μL.
Thermal cycling conditions: 95oC (10 min); [95oC (40 sec), 56oC (30 sec), 72oC (2 min)]35X, 72oC (7 min)
HO
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x
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x
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x
x
Random
decamer
Oligo(dT) +
Random
decamer
Figure 10
Triplex One-step RT-PCR amplification using CleanAmp™
Precision Primers
Unmodified
CleanAmp™
Precision
30.0
20.0
10.0
Brain
Thymus
Trachea
Total RNA Tissue Source
PCR conditions: 1X PCR buffer (20 mM Tris (pH 8.4), 50 mM KCl), 1.5 mM MgCl2, Primers (0.5 μM), polydT18
primer (1 uM), 0.16 mM dNTPs, 0.5 μg Human trachea total RNA, 50 U MMLV reverse transcriptase, 0.1 PM
TM probe, 0.625 U Taq DNA polymerase, 50 μl.
Thermal cycling conditions:
42oC (40 min); 95oC (10 min); [95oC (30 sec), 60oC (30 sec), 72oC (30 sec)] 30X; 72oC (5 min).
Failure
Turbo Primers effectively amplify all three targets
over a wide range of primer concentrations.
Unmodified
Primers
P O
O
x
x
Precision Primers demonstrate greatest amplicon yield in
multiplex One-Step RT-PCR relative to unmodified primers.
PPi
Enzyme
e
x
x
40.0
B1
HO
X
x
Precision Primers succesfully amplify three targets individually and
simultaneously using different priming techniques.
143 bp
114 bp
82 bp
61 bp
d e f g h i
5'
Unmodified
Primers
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1
a b c d e f g h i
CleanAmp™
Primers
P O
O
O
“Hot Start”
Activation
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B2
O
X O
Target
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x
0.5
1
O
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0.1
0.1
5'
x
600bp. amplicon
0.1
ABCA7
ABCA6
ABCA5
ABCC10
533bp. amplicon
0.1
CleanAmp™
Primers
X
x
x
x
PCR conditions: 1X PCR buffer (20 mM Tris (pH 8.4), 50 mM KCl), 1.5 mM MgCl2, Primers (0.5 μM), polydT18
primer (1 uM), 0.16 mM dNTPs, 0.5 μg Human trachea total RNA, 50 U MMLV reverse transcriptase, 0.1 PM
hydrolysis probe, 0.625 U Taq DNA polymerase, 50 μl.
Thermal cycling conditions:
42oC (40 min); 95oC (10 min); [95oC (30 sec), 60oC (30 sec), 72oC (30 sec)] 30X; 72oC (5 min).
Singleplex and fourplex One-Step RT-PCR amplification
using CleanAmp™ Precision Primers
Limited multiplex optimization with CleanAmp™
Turbo Primers
0.5
x
Oligo(dT)
Figure 7
Figure 4
533bp. amplicon
Figure 1
x
x
x
NTC
1X PCR buffer (20 mM Tris (pH 8.4), 90 mM KCl, 2.5 mM MgCl2), Primers (0.2 μM), 0.4 mM dNTPs,
5,000 copies Lambda gDNA, 2.5 U Taq DNA polymerase, 50 μL.
Unmodified
Proposed activation mechanism of CleanAmp™ Primers
x
x
114 bp
82 bp
Number of
Targets
PCR conditions:
1X PCR buffer (20 mM Tris (pH 8.4), 50 mM KCl, 2.5 mM MgCl2), Turbo** CleanAmpTM Primers (0.2
μM), hydrolysis probe (0.1 μM), 0.2 mM dNTPs, -50 50,000 copies Lambda gDNA, 1.25 U Taq DNA
polymerase, 25 μL; reactions performed in triplicate.
primer
concentration
(PM)
x
x
143 bp
Amount of template (copies)
Cq (dRn)
allows for an efficient one-step RT-PCR reaction that provides high specificity
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x
1.E+06
transcriptase PCR (RT-PCR), the doubly-modified primers have been proven
to be the optimal choice. The presence of two thermolabile protecting groups
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ABCA7
either one or two thermolabile 4-oxo-tetradecyl (OXT) modifications to prevent
singly-modified primers provide greater amplification efficiency, specificity,
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Turbo**
15
1.E+01
to multiplex PCR, we evaluated Hot Start modified primers which contain
released after a Hot Start activation step. Herein, we find that the
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Unmodified
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DNA polymerase extension at low-stringent temperatures, and that are
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962 bp
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problematic targets extremely difficult. To improve upon the problems specific
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Amount of template (copies)
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distribution of amplicon products, making quantification and detection of
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ABCA6
1.E+02
Amount of template (copies)
Furthermore, preferential amplification of certain targets leads to an unequal
x
ABCA5
15
1.E+01
1.E+06
Unmodified
Turbo
Turbo**
Figure 8
Triplex One-Step RT-PCR amplification using CleanAmp™
Precision Primers
ABCA5 singleplex
ABCA6 singleplex
ABCA7 singleplex
ABCA5 triplex
ABCA6 triplex
ABCA7 triplex
Precision Primers allow for reliable quantification of three targets
individually and simultaneously in different tissues.
PCR conditions: 1X PCR buffer (20 mM Tris (pH 8.4), 50 mM KCl), 1.5 mM MgCl2, Primers (0.5 μM), polydT18
primer (1 uM), 0.16 mM dNTPs, 0.5 μg Human trachea total RNA, 50 U MMLV reverse transcriptase, 0.1 PM
hydrolysis probe, 0.625 U Taq DNA polymerase, 50 μl.
Thermal cycling conditions:
42oC (40 min); 95oC (10 min); [95oC (30 sec), 60oC (30 sec), 72oC (30 sec)] 30X; 72oC (5 min).
X = Thermolabile protecting group
Figure 5
Figure 2
960 bp
600 bp Triplex
Tem plate Targets
Unmodified
Turbo**
Fluorescence (dRn)
1) CleanAmp™ Turbo Primers give optimal performance for multiplex
amplification of DNA targets reducing optimization time.
2) Turbo Primers provide efficient amplification of up to nine targets ranging in
size.
20
0
40
20
40
NTC
1 ng
5 ng
533 bp
962 bp 600 bp Triplex
Turbo Primers demonstrate greatest amplicon yield in
multiplex PCR relative to unmodified primers.
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250 ng
ABCA5 : Eff. = 96.8%
Y = -3.402*LOG(X) + 39.08
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ABCA6 : Eff. = 97.8%
Y = -3.377*LOG(X) + 35.07
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* Fraction of amplicon formation is
normalized to Turbo
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3) In multiplex Real-time PCR, Turbo Primers improve the limit of detection.
50 ng
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*denotes
polymerases
with Hot Start
activation
0
Cycles
Cycles
Cq (dRn)
533 bp
Conclusion
ABCA6
(Cy5 detection)
1.35
1.15
0.95
0.75
0.55
0.35
0.15
-0.05
0
Hot Start pol. D*
962bp
600bp
533bp
Hot Start pol. C*
x
Hot Start pol. B*
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Hot Start pol. A*
x
x
Turbo** primers + Taq
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Hot Start pol. D*
x
x
Hot Start pol. C*
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Unmodified primers + Taq
x
x
Hot Start pol. B*
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Hot Start pol. A*
x
Turbo**
1.2
1
0.8
0.6
0.4
0.2
0
Turbo** primers + Taq
Unmodified primers + Taq
Unmodified
Amplicon Yield
Utility of CleanAmp™ Turbo Primers in singleplex and multiplex PCR
ABCA7
(HEX detection)
ABCA5
(FAM detection)
Hot Start technologies evaluation
0.1
5,000 copies
Lambda DNA
1
FAM - ABCA5
50,000 copies
Lambda DNA
10
CY5 - ABCA6
100
1000
ABCA7 : Eff. = 104.3%
Y = -3.224*LOG(X) + 37.58
4) Precision Primers allow for both the RT and PCR steps of RT-PCR to be
combined into a single reaction set-up without sacrificing specificity.
5) In one-step RT-PCR, Precision Primers can be used with different reverse
transcription primers.
6) Triplex Real-time One-Step RT-PCR amplification in different tissues was
possible with Precision Primers.
HEX - ABCA7
Acknowledgements
Human Liver total RNA (ng)
Turbo Primers amplify all three targets efficiently compared to
other Hot Start polymerases.
The use of Precision Primers in Real-time One-Step RT-PCR
allows a wide range of linearity for input RNA.
Jonathan Shum, Joyclyn Yee, Terry Beck, Judy Le, Angela Tenenini, Brianne Grajkowski,
Olga Adelfinskaya, Dave Combs, Stephanie Perry, Katelyn Hanesana, Paul Imperial, Carina
Ly, Kristin Aquino, Bryan Murphy, Jennifer Magowan.
NIH grants: 1R43GM072177-01A1, 2R44GM072177-02, and 5R44GM072177-03
1X PCR buffer (20 mM Tris (pH 8.4), 50 mM KCl, 2.5 mM MgCl2), Primers (0.5 μM), 0.2
mM dNTPs, 50,000 copies Lambda gDNA, 1.25 U Taq DNA polymerase, 50 μL.
Thermal cycling conditions:
95oC (10 min); [95oC (40 sec), 56oC (30 sec), 72oC (2 min)]35X, 72oC (7 min)
PCR conditions: 1X PCR buffer (20 mM Tris (pH 8.4), 50 mM KCl, 2.5 mM MgCl2), Primers (0.075 PM), 0.2
mM dNTPs, 5 copies HIV-1 gDNA, DNA polymerases: 1.25 units of Taq and Hot Start DNA polymerases (A-D),
Thermal cycling conditions: 95oC (10 min); [95oC (40 sec), 56oC (30 sec), 72oC (1 min)] 35X, 72oC (7 min)
PCR conditions: 1X PCR buffer (20 mM Tris (pH 8.4), 50 mM KCl), 1.5 mM MgCl2, Primers (0.5 μM), polydT18
primer (1 uM), 0.16 mM dNTPs, 1, 5, 10, 50, and 250 ng Human trachea total RNA, 50 U MMLV reverse
transcriptase, 0.1 mM hydrolysis probe, 0.625 U Taq DNA polymerase, 50 μl.
Thermal cycling conditions:
42oC (40 min); 95oC (10 min); [95oC (30 sec), 60oC (30 sec), 72oC (30 sec)] 30X; 72oC (5 min).
** Denotes CleanAmpTM Primers containing 4-oxo-pentyl modifications
For further information, please contact Natasha Paul, [email protected]
The Modified Nucleic Acid Experts
9955 Mesa Rim Road
|
San Diego, CA 92121
|
800-863-6801
|
www.trilinkbiotech.com
|
[email protected]