Download CleanAmp™ Hot Start dNTPs

The Universal Hot Start Tool: CleanAmp™
Hot Start dNTPs
The Modified Nucleic Acid Experts
Sabrina Shore, Jordana Henderson, Anton McCaffrey, Gerald Zon, Alexandre Lebedev, Richard Hogrefe
TriLink BioTechnologies, Inc., San Diego, CA
26000
26000
30000
E4, ABCA7_5ng, FAM30000
E5, ApoB_50ng, FAM
28000
28000
E6, ApoB_5ng, FAM
26000
26000
38000
38000
D3, ABCA7_50ng, 36000
FAM
36000
D4, ABCA7_5ng, FAM
34000
D5, ApoB_50ng, 34000
FAM
D6, ApoB_5ng, FAM
32000
32000
E3, ABCA7_50ng, FAM
30000
E4, ABCA7_5ng,30000
FAM
E5, ApoB_50ng, FAM
28000
28000
E6, ApoB_5ng, FAM
26000
26000
24000
24000
24000
24000
24000
24000
34000
34000
32000
32000
30000
30000
28000
28000
22000
22000
20000
20000
18000
18000
16000
16000
14000
14000
12000
12000
B4GN4
78% GC
720 bp
Reference
Sequence
P
O
P
P
HO
O
O
O
O
O
OH
Activation
OH
H
O
O
P
P
P
HO
O
O
O
H
O
O
OH
O
8000
8000
6000
6000
6000
6000
4000
4000
4000
4000
4000
4000
2000
2000
2000
2000
00
00
2000
2000
00
Input RNA
ABCA7
Mean Cq
Cycle
Cycle
Threshold:
ApoB
Mean Cq
841 (Adjusted manually)
Input RNA
ABCA7
Mean Cq
Threshold:
ApoB
Mean Cq
841 (Adjusted manually)
Input RNA
ABCA7
Mean Cq
ApoB
Mean Cq
Baseline settings: automatic, Drift correction ON
Baseline settings: manual range: 3 ... 15, Drift correction ON
50 ng
25.54
17.58
50 ng
28.49
21.55
50 ng
26.69
20.38
5 ng
28.79
21.02
5 ng
31.79
26.58
5 ng
30.26
24.31
ABCA5
Figure 5: PCR Amplification Improves
Sequencing Accuracy of Homopolymer
Regions and Allows for Longer Reads
450
460
A
470
480
B
C
D
E
F
ACleanAmp™ One-Step RT-PCR Master Mix
BPath-ID™ Multiplex One-Step RT-PCR Kit (Life Technologies)
C Verso 1-Step RT-PCR Kit with Thermo-Start Taq (Thermo Scientific)
DSuperscript® III One-Step RT-PCR System with Platinum Taq (Life Technologies)
EiTaq® Universal Probes One-Step Kit (Bio-rad)
FOneTaq® RT-PCR Kit (New England Biolabs)
490
Figure 10: One-Step RT-PCR with
CleanAmp™ dUTP and UNG Prevents
Carry Over Contamination
Base
DIPA Reference Sequence (819 bp, 73% GC)
100
200
300
400
500
600
700
789
440
0%
Phred Score (Sequency Quality)
<20 (low) 20-40 (medium)
Standard 7-deaza-dGTP dNTP Mix
CleanAmp™ 7-deaza-dNTP Mix
450
460
Step up new PCR
reaction
Minor
contamination
from previous
PCR degraded by
UNG (25°C)
RT Step &
UNG inactivated
(47°C)
PCR using
dUTP
3000
ABCA7 Target
(Trachea Total RNA)
ABCA7 Target +
dU contaminant
dU contaminant
dU contaminant
with UNG
>40 (high)
2000
1000
CleanAmp™ dNTPs Compatible with
a Variety of Reverse Transcriptases
Target (264 bp)
Standard dNTPs
CleanAmp™ dNTPs
Hot Start DNA Polymerase
Superscript® III
− + −
− + +
M-MLV
(RNase H-)
Superscript® II
Hot Start RT
Hot Start PCR
Figure 7: Quantification of RNA
Expression Levels by Real-Time Hot Start
One-Step RT-PCR
28000
26000
24000
22000
20000
20000
18000
16000
14000
12000
10000
10000
8000
6000
4000
2000
00
Fluorescence (norm)
Fluorescence (nm)
30000
30000
0
0
2
4
6
8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
10
20
30
40
Cycle
Cycle
Threshold:
971 (Noiseband)
Baseline settings: automatic, Drift correction ON
0
0
10
20
Cycles
30
40
Conclusion
• CleanAmp™ dNTPs are compatible with a variety of enzymes
and molecular biology assays.
• CleanAmp™ dNTPs enhance the specificity of multiplex
PCR where amplification of at least 50 targets has been
demonstrated (data not shown).
Side Product
Fragile X
>85%
~300 bp
PCR Crosscontamination
95% GC Region
50%
Mis-priming
Target (264 bp)
Figure 3: Improved Amplification Yield
and Specificity for Targets Up to 85%
GC-Content
PCR Product
with dUTP from
previous reaction
100%
RT and PCR Steps Benefit
from Hot Start Control
50 base pair ladder
Standard dNTP Mix
7-deaza-dGTP Mix
CleanAmp™ 7-deaza-dNTP Mix
841 (Noiseband)
00 22 44 66 8 10
10 12
12 14 16 18
18 20
20 22 24
24 26 28
28 30 32
32 34
34 36 38 40
40
Cycle
0 22 44 66 88 10
10 12
12 14
14 16
16 18
18 20
20 22
22 24
24 26
26 28 30
30 32 34
34 36 38
38 40
40
Cycle
ABCA1
ABCA7
Figure 6: One-Step RT-PCR Specificity is
Improved by Hot Start Activation of the
RT and PCR Steps
IGFR2
80%
678 bp
12000
12000
PBGD
GC Content
O
BRAF
75%
185 bp
14000
14000
Figure 9: CleanAmp™ One-Step RT-PCR
Master Mix Successfully Amplifies Four
Targets in Multiplex RT-PCR
50 base pair ladder
Standard 7-deaza-dGTP dNTP Mix
CleanAmp™ 7-deaza-dNTP Mix
HO
Figure 2: CleanAmp™ dNTPs Improve
the Performance of DNA Polymerases
AGT
60%
242 bp
16000
16000
6000
6000
Threshold:
95.2% High
Quality
18000
18000
OH
Activation occurs with a temperature increase or pH decrease. CleanAmp™ dNTPs
deprotect with a t½ of 5 min at 95°C in PCR buffer.
Target
GC-Content
Length
12000
12000
20000
20000
10000
10000
34.4% High
Quality
1
CleanAmp™ Modification:
3'-Tert-Butoxy-Ethoxy
14000
14000
Cycle
O
O
16000
16000
0 22 44 66 88 10
10 12
12 14
14 16
16 18
18 20
20 22
22 24
24 26
26 28
28 30
30 32
32 34
34 36
36 38
38 40
40
Cycle
Figure 1: CleanAmp™ Hot Start dNTP
Structure & Activation
Base
18000
18000
22000
22000
8000
8000
Baseline settings: manual range: 3 ... 15, Drift correction ON
440
O
20000
20000
8000
8000
B4GN4 Target (340-394 bp homopolymer repeat region with 89.1% GC)
O
22000
22000
10000
10000
10000
10000
D3, ABCA7_50ng, FAM
D4, ABCA7_5ng, FAM
D5, ApoB_50ng, FAM
D6, ApoB_5ng, FAM
E3, ABCA7_50ng, FAM
E4, ABCA7_5ng, FAM
E5, ApoB_50ng, FAM
E6, ApoB_5ng, FAM
Fluorescence (norm)
36000
36000
Fluorescence (norm)
38000
38000
D3, ABCA7_50ng, FAM 36000
36000
D4, ABCA7_5ng, FAM
34000
D5, ApoB_50ng, FAM 34000
D6, ApoB_5ng, FAM
32000
32000
E3, ABCA7_50ng, FAM
38000
38000
Sequence
Analysis
LeGene Premium 1-Step
RT-PCR Master Mix
Fluorescence (norm)
PCR
Clean-Up
EMD One Step RT-PCR
Master Mix Kit
Fluorescence (norm)
PCR
Amplification
Sanger
Dideoxy
Sequencing
CleanAmp™ One-Step RT-PCR
Master Mix (Cat # L-5104)
Fluorescence (norm)
PCR is a powerful tool which is being consistently refined to push
the limits of speed, sensitivity, and specificity. A Hot Start method is
imperative in any PCR technique to improve performance by reducing
primer dimers and to prevent mispriming. Many Hot Start technologies
are limited to specific assay types or by higher temperature
requirements for activation. In contrast, Hot Start dNTPs are a universal
approach that offers a flexible mode of activation which relies on a
combination of temperature, pH, and time. A thermolabile protecting
group inhibits dNTP incorporation at ambient temperatures when
many side reactions occur and is gradually released as temperature
is elevated or pH is lowered. Hot Start dNTPs have demonstrated that
they improve the performance of most reverse transcriptase and
DNA polymerase enzymes by increasing yields and specificity in
various assay types. The CleanAmp™ thermolabile protecting group
can be applied to dNTP analogs such as 7-deaza-dGTP and dUTP.
CleanAmp™ 7-deaza-dGTP improves GC-rich target amplification for
targets up to 85% GC-rich and allows for more accurate and longer
reads for downstream Sanger dideoxy sequencing. CleanAmp™ dUTP
is used in carry over prevention assays in conjunction with Cod UNG
(Uracil-DNA Glycosylase). CleanAmp™ dNTPs are an ideal tool for RTPCR where they provide Hot Start functionality for both RT and PCR
steps. At standard RT temperatures (45-65°C) only a fraction of dNTPs
are available for cDNA synthesis which limits the ability of off target
reactions from occurring. Once PCR temperatures (95°C) are reached,
an abundance of dNTPs are available for efficient amplification. In RTPCR assays Hot Start dNTPs exhibit improved yield and specificity when
compared to other RT-PCR kits and can effectively amplify multiple
targets at once. CleanAmp™ dNTPs are a universal Hot Start tool that
can be integrated into a variety of assays to enhance performance.
Figure 8: CleanAmp™ One-Step RT-PCR
Generates Lower Cq Values
Fluorescence (norm)
Abstract
Figure 4: PCR Amplification with
CleanAmp™ 7-deaza-dGTP Improves
Accuracy of Dideoxy Sequencing in
GC-Rich Targets
C3, Std_500/1, FAM
C4, Std_100/1, FAM
C5, Std_20/1, FAM
C6, Std_4/1, FAM
C7,
Std_0.8/1,
Liver
RNAFAM
(ng)
C8, Std_0.16/1, FAM
500
C9, Std_0.032/1,
FAM
C10, NTC, FAM
100
D3, Std_500/2, FAM
D4, Std_100/2,
FAM
20
D5, Std_20/2, FAM
4
D6, Std_4/2,
FAM
D7, Std_0.8/2, FAM
0.8
D8, Std_0.16/2, FAM
D9, Std_0.032/2,
FAM
0.16
E3, Std_500/3, FAM
0.032 FAM
E4, Std_100/3,
E5, Std_20/3, FAM
NYC
E6, Std_4/3, FAM
E7, Std_0.8/3, FAM
E8, Std_0.16/3, FAM
• CleanAmp™ dNTPs can be employed in fast PCR cycling
protocols to achieve high quality data in as little as 24 minutes
(data not shown).
• CleanAmp™ 7-deaza-dGTP provides robust amplification and
sequencing of GC-rich targets.
• CleanAmp™ 7-deaza-dGTP improves sequencing of GC-rich
targets and allows longer sequence reads.
• CleanAmp™ dNTPs improve one-step RT-PCR specificity by
introducing Hot Start control to both the RT and the PCR steps.
• CleanAmp™ One-Step RT-PCR Master Mix outperforms other
commercial RT-PCR kits and master mixes.
• CleanAmp™ dUTP with UNG protects against PCR crosscontamination.
NIH grants: 1R43GM079836-01, 2R44GM079836-02 and 5R44GM079836-03
Reaction conditions can be found at www.trilinkbiotech.com/posters
Contact: Sabrina Shore, [email protected]
The Modified Nucleic Acid Experts
www.trilinkbiotech.com
™