yong you ms, associate product manager integrated dna...
TRANSCRIPT
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Integrated DNA Technologies
PrimeTime and qPCR
Yong You MS, Associate Product Manager
IDT Confidential: This document may contain confidential and proprietary information. Any unauthorized review, use, disclosure or distribution is prohibited
Webinar for Hong Kong University, March 29th, 2011
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Overview
Basics of qPCR
SYBR vs Hydrolysis Probes
PrimeTime vs MGB TaqMan Assays
IDT Pre-designed PrimeTime Assays
IDT ZEN Double Quenched Probes
IDT SciTools
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BASICS OF qPCR
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Real Time quantitative PCR
• The Polymerase Chain Reaction (PCR) technique was invented in 1985 by Dr. Kary B. Mullis
• Real Time PCR was Developed by Perkin Elmer in 1995.
• Mechanism: Fluorescent signal increase proportionally to the amplicon increase.
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Difference between traditional PCR and qPCR
PCR - Gel based analysis at the end of amplification in the plateau phase
qPCR - Fluorescent measurements during the entire amplification cycle
qPCR is THE method of choice for quantitative gene expression analysis
Real TimeNo. of DNA molecules 2500 500 100 20
4
Traditional PCR detection
RT-PCR
Threshold
Cycle No. 15 20 25 30 35
Plateau
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Results of a qPCR assay
• Cycle number (Cq) reflects number of copies of starting template DNA
Early cycle number -----more DNA
Late cycle number -----less DNA
*
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• Fluorescence is a property of a class of molecules
• Absorb light energy at a short wavelength
• Release this light energy at a longer wavelength
• Length of time between absorption and emission is 10-8 to 10-9s
SO
S1’
S1
hνex hνem
1
2
3
Brought to you by the phenomenon of
Fluorescence!
Release of energy as fluorescence
Fluorescence (熒光現象)
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Types of qPCR
1 2
3 4
1 2
1 2 31 2
Intercalators
(Sybr Green)
Hybridization FRET
Molecular Beacon5’ Nuclease assay,
Prime Time Assay,
Taqman assay
cgctacc
Fam
494/518 IablkFqZ
SYBR is a fluorescent dye not linked to a sequence,all other probes have the fluorescent dye attached to certain nucleic acid
熒光染料 : SYBR Green, EvaGreen
熒光探針 : FRET( 熒光共振能量轉移 )Molecular Beacon(分子信標)
5’ Nuclease Assay
( 5’ 水解酶分析技術)
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5’ nuclease qPCR assay
5’ 3’3’ 5’
a c g a c g a
3’ 5’Primer/oligo
t c g a t c g a t c g a t a g g c c a t c g c g a c t c c t t g c t g c t
gctagct
5’ 3’Primer/oligo
gcta ccc
R Q
aa ta gct
a g c t a g c t a g c t a t c c g g t a g c g c t g
3’ 5’
a g g a a c g a g ac
t tt tt tt t t
12
3
95C
65C
During amplification, the 5‘ to 3’ exonuclease activity of polymerase will hydrolyze the probe, separating the fluorophorfrom the quencher, the detection system will detect fluorescence signal. Each cycle, more DNA is made, more signal is generated. This is the basis of qPCR quantification.
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PCR amplification and copy number
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Real-time qPCR general design rules
• Good primer and probe design
• Check qPCR instruments compatibility with dyes
• Use a hot-start, high-fidelity enzyme
• Additional considerations for multiplex experiments• Especially in assay design and choices of dyes
• Determine controls (both positive and negative)
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Instrument Compatibility with Reporter
Dyes
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Multiplex qPCR
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Primer and Probe design criteria for PrimeTime
Primers
equal Tm (60-64 C)
18-30 bases in length
no runs of consecutive Gs
Probes
Tm value 6-10C higher than primers
no longer than 30-35 bases
no runs of consecutive Gs
No Gs on the 5’ end
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Designed to limit genomic DNA amplification—How?
Exon Exon Exon Exon1 2 3 4
intron intron(1000bp) intron
DNA
1 2 3 4RNA(immature)
Transcription
Splicing
(amplicon is large)
• Assays are designed to span exons
• Exon – Exon junctions maybe targeted
• Primers could span an intron greater than 1 kb
(amplicon is small and specific)
Mature RNA1 2 43
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IDT SciTools
IDT offers a variety of design and analysis tools. The SciTools suite includes tools for designing assay components as well as determining the properties of any sequence entered.
OligoAnalyzerProvides accurate analysis of melting temperature
PrimerQuestCustom design of primers and probes for PCR and qPCR reactions
UNAFoldProvides analysis of secondary structures
RealTime PCR DesignqPCR assay design based on exon boundaries
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IDT SciTools
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SciTools – RealTime PCR
IDT recommends using the RealTime PCR design tool located on the IDT website to ensure all of the important parameters will be included in the assaydesign.
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SYBR GREEN VS HYDROLYSIS PROBES
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Real Time qPCR Method of Detection
cDNA+
Water, Buffer, Primers, dNTPs, Enzyme, MgCl2 (MASTERMIX)+
Method of detection
cgctacc
R Q
Probe (PrimeTime Assays)
R= reporter/fluorophore,Q=quencher
PrimeTime assays have adual labeled probeand is a superior method of detection
SYBR Green OR(fluorescentIntercalating dye)
SYBR – non-sequence specific
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550CPrimers anneal, dsDNASYBR Green intercalates and fluoresces
720CAs more dsDNA is synthesizedIncreased fluorescence is observed
940CDNA is denatured to ssDNA formNo SYBR green binding
(SYBR Green only intercalates with dsDNA)
Method of detection: SYBR Green (494nm/520nm)
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SYBR Green Summary
Advantages: Inexpensive (as do not require a dye-labeled probe)
General screening prior to moving to probe based assays
OK when the target is abundant
Disadvantages: Not sequence specific: bind to primer-dimers and incorrect amplicon
Requires melting curve analysis
Cannot perform multiplex reaction
Cannot be used for Allelic Discrimination
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PRIMETIME VS TAQMAN MGB ASSAYS
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IDT PrimeTime qPCR Products
PrimeTimeTM qPCR Assays Gene expression PrimeTime Pre-designed Assays PrimeTime qPCR Assays
PrimeTimeTM qPCR Probes Gene Expression Copy Number Estimation
PrimeTimeTM LNA Probes Genotyping
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Components of a PrimeTimeTM Assay
Each PrimeTimeTM Assay consists of two PCR primers and a dual labeled PrimeTimeTM probePre-mixed in a tube primer probe ratio = 2:1(Mini), 1:1 (Std) and 4:1 (XL)
gctagct
5’ 3’Primer/oligo
a c g a c g a
3’ 5’Primer/oligo
cgctacc
R Q
Probe
R= reporterQ=quencher
+Z
PrimeTime Assay include IDT’s proprietary ZEN Double Quenched Probes (DQP).The DQPs are linear probes, in addition to the 5’ fluorophor and 3’ quencher, they also containan internal ZEN quencher.
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PrimeTime Assays
5' Dye 3' Quencher Mini Standard XL
FAM ZEN/Iowa Black FQ* • • •
FAM TAMRA • •
HEX Iowa Black FQ • •
TET Iowa Black FQ • •
Cy5 Iowa Black RQ • •
Available Dye and Quencher Combinations for PrimeTime qPCR AssaysPrimer/probe ratios from 1:1 to 4:1(primer concentration varies, probe is fixed)
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PrimeTime Assay Performance = Gold Standard Performance
Efficiency 101.1%Std Curve R2 0.9996
Gene CCL5Copies shown 10 to 1E7Master Mix ABI MMxInstrument ABI7900
NTC
10 copies
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First 4 assays run on ABI machine, the Cy5 assay is run on theRoche LC480
Performance of Assays with different dye /quencher
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0 Freeze - Thaws 15 Freeze - Thaws 30 Freeze - Thaws
Stability Test for PrimeTime Assays
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PrimeTimeTM for gene expression
PrimeTime qPCR probes and assays can be used to study gene expression.
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product.
Protein-coding genes
Non-protein coding genes:ribosomal RNA (rRNA) genes transfer RNA (tRNA) genes
proteins
functional RNA
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PrimeTime qPCR for Copy Number Estimation
Copy Number Estimation – using techniques such as real time qPCR to estimate the copy number of certain genes of interest, this will require an internal standard
Copy-number variations (CNVs): Alterations of genomic DNA that have been deleted or amplified on certain chromosomes
PrimeTime qPCR probes can be used to study Copy Number Variations.
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PrimeTimeTM LNA probes for Genotyping
Genotyping (Allelic discrimination): determining the genotype of an individual by examining its DNA sequence using molecular techniques
The Allelic Discrimination assay detects:• Homozygotes
(samples having only allele 1 or allele 2)• Heterozygotes
(samples having both allele 1 and allele 2)
PrimeTime qPCR LNA probes can be used to study allelic discrimination.
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Allelic discrimination assay
• Multiplexed assay to detect variants of a single nucleic polymorphism (SNP)
For each sample in an AD assay, a unique pair of probes is used, for example, two PrimeTime LNA probes that target the same SNP site. • 1 probe is a perfect match to the wild type (allele 1) • 1 probe is a perfect match to the mutation (allele 2)
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TaqMan MGB probes by ABI (MGB = Minor groove binder)
These probes are sold exclusively by ABI because they have the license for MGB
• MGB clamps the probe and stabilizes the probe onto the target
• MGB raises the Tm by as much as 10oC
• Probes can be shorter than traditional dual labeled probes, but still maintain Tm.
The MGB is attached in addition to the standard fluorophore reporter such as FAM and quencher
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If MGB probe sequences were used for PrimeTime…
Probe Tm has to be 6 to 10oC higher than primers
Please lengthen the probe flanking sequence to increase Tm
Length of MGB probes likely less than 18 bpWithout MGB Tm is going to be low at 45-54oC(Please check with IDT OligoAnalyzer, Mg2+ 3 mM, dNTPs 0.8mM)
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How to find PrimeTime products on IDT website?
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IDT PRE-DESIGNED PRIMETIME ASSAYS
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Introducing PrimeTime Pre-designed qPCR Assays
Pre-designed assays are available for human, mouse and rat Designed to span every exon boundary in the transcriptome
Current coverage rate for human is 97.4% of exon junctions
Assays are designed to Avoid SNPs locations
Eliminate non-specific amplification
Reduce impact from secondary structure formation
Simple ordering tool Filter to quickly view common or transcript specific assays
Sort by recommended assays
Select reaction size, dye-quencher, or primer to probe ratio
Group by species, gene symbol or refseq number for quick assay selection
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Designing successful qPCR assays
1. Up-to-date transcript sequence information, including: exon boundaries
splice variants
SNP locations
2. Design of primers and probes, including: accurate melting temperature
elimination of non-specific amplicon generation
reduction of secondary structure formation
3. High quality oligos that reduce impurities and provide reproducible results over time
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Transcript information is changing rapidly
Assay performance heavily relies on the quality of sequence information you are trying to target.
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# o
f SN
Ps
Number of SNPs in dbSNP
Identified
Validated
Next Generation Sequencing has significantly increased the number of SNPs and splice variants identified. Having up to date sequence information is critical to qPCR assay performance
April 200815M SNPs
Sept 201030M SNPs
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IDT has expertise in oligo thermodynamics
For over ten years, IDT has dedicated a team to understanding oligo thermodynamics. With thousands of melt curves performed, IDT oligo melting temperature calculations are estimated to be the most accurate in the industry.
IDT continues innovative DNA thermodynamic research and integrates those improvements into its calculation and oligonucleotide design tools
Publications:Owczarzy R, Tataurov AV, Wu Y, Manthey JA, McQuisten KA, Almabrazi HG, Pedersen KF, Lin Y, Garretson J, McEntaggart NO, Sailor CA, Dawson RB and Peek AS
(2008) IDT SciTools: a suite for analysis and design of nucleic acid oligomers. Nucleic Acids Res., 36:163-169.
Owczarzy R, Moreira BG, You Y, Behlke MA, Walder JA (2008) Predicting stability of DNA duplexes in solutions containing magnesium and monovalent cations. Biochemistry, 47: 5336-5353. Additional data
Tataurov AV, You Y, Owczarzy R (2008) Predicting ultraviolet spectrum of single stranded and double stranded deoxyribonucleic acids. Biophys. Chem., 133: 66-70.
You Y, Moreira BG, Behlke MA, Owczarzy R (2006) Design of LNA probes that improve mismatch discrimination. Nucleic Acids Res., 8: 60.
Owczarzy R (2005) Melting temperatures of nucleic acids: Discrepancies in analysis. Biophys. Chem., 117: 207-215.
Moreira BG, You Y, Behlke MA, and Owczarzy R (2005) Effects of fluorescent dyes, quenchers, and dangling ends on DNA duplex stability. Biochem. Biophys. Res. Comm., 327: 473-484
Owczarzy R, You Y, Moreira B, Manthey JA, Huang L, Behlke MA, and Walder JA (2004) Effects of sodium ions on DNA duplex oligomers: Improved predictions of melting temperatures. Biochemistry, 43: 3537-3554
Owczarzy R, Dunietz I, Behlke MA, Klotz IM, and Walder JA (2003) Thermodynamic treatment of oligonucleotide duplex-simplex equilibria. Proc. Natl. Acad. Sci. USA, 100: 14840-14845
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IDT has expertise in oligo thermodynamics
For over ten years, IDT has dedicated a team to understanding oligo thermodynamics. With thousands of melt curves performed, IDT oligo melting temperature calculations are estimated to be the most accurate in the industry.
IDT continues innovative DNA thermodynamic research and integrates those improvements into its calculation and oligonucleotide design tools
Publications:Owczarzy R, Tataurov AV, Wu Y, Manthey JA, McQuisten KA, Almabrazi HG, Pedersen KF, Lin Y, Garretson J, McEntaggart NO, Sailor CA, Dawson RB and Peek AS
(2008) IDT SciTools: a suite for analysis and design of nucleic acid oligomers. Nucleic Acids Res., 36:163-169.
Owczarzy R, Moreira BG, You Y, Behlke MA, Walder JA (2008) Predicting stability of DNA duplexes in solutions containing magnesium and monovalent cations. Biochemistry, 47: 5336-5353. Additional data
Tataurov AV, You Y, Owczarzy R (2008) Predicting ultraviolet spectrum of single stranded and double stranded deoxyribonucleic acids. Biophys. Chem., 133: 66-70.
You Y, Moreira BG, Behlke MA, Owczarzy R (2006) Design of LNA probes that improve mismatch discrimination. Nucleic Acids Res., 8: 60.
Owczarzy R (2005) Melting temperatures of nucleic acids: Discrepancies in analysis. Biophys. Chem., 117: 207-215.
Moreira BG, You Y, Behlke MA, and Owczarzy R (2005) Effects of fluorescent dyes, quenchers, and dangling ends on DNA duplex stability. Biochem. Biophys. Res. Comm., 327: 473-484
Owczarzy R, You Y, Moreira B, Manthey JA, Huang L, Behlke MA, and Walder JA (2004) Effects of sodium ions on DNA duplex oligomers: Improved predictions of melting temperatures. Biochemistry, 43: 3537-3554
Owczarzy R, Dunietz I, Behlke MA, Klotz IM, and Walder JA (2003) Thermodynamic treatment of oligonucleotide duplex-simplex equilibria. Proc. Natl. Acad. Sci. USA, 100: 14840-14845
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Pre-designed Validation Results
Validation Highlights
More than 99% of assays exhibited >90% efficiency
100% of the assays had R2 values >0.99
Mean efficiency across all 150 assays was 94.5%
Over 2/3rds of the assays had efficiencies >94%
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Example Dilution Series Results
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Comparison to competitor pre-designed assays
25 assays from Competitor A were compared to an equal number of IDT’s PrimeTime Pre-designed qPCR Assays.
The Competitor A assays consisted of 15 inventoried pre-designed assays and 10 made-to-order pre-designed assays.
To ensure an accurate comparison was made, the PrimeTime Assays and Competitor A assays were selected to span the same exon boundary of each gene.
The reactions were run with the Applied Biosystems Gene Expression Master Mix and identical thresholds were set for all runs
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qPCR Efficiency
PrimeTime Pre-designed qPCR Assays Have Higher qPCR Efficiency and a Smaller Distribution Range than Competitor A Assays.
PrimeTime qPCR Assays were compared to matched Competitor A assays using 5-fold dilutions of cDNA and the Applied Biosystems TaqMan™ Gene Expression Master Mix. The reactions were run on the ABI 7900HT Fast Real-Time PCR System with the following PCR cycling conditions: 2 min. 50°C;
10 min. 95°C; 45 x (15 sec. 95°C, 1 min. 60°C). Identical thresholds were set for all runs for comparison across assays.
0
2
4
6
8
10
12
14
88 - 90 90 - 92 92 - 94 94 - 96 96 - 98 98 - 100
Fre
qu
en
cy
qPCR Efficency (%)
Comparison of qPCR Efficiency
PrimeTime
Competitor A
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Example qPCR curves
PrimeTime Competitor A
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Example curves pre-baselined
0
1
2
3
4
5
6
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
Rn
(F
AM
/RO
X)
Cycle Number
Comparison of Assay Performance with WDR3 (NM_006784)
■ PrimeTime Assays
■ Competitor A
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PrimeTime Cq’s are nearly 1 full Cq lower than Comp. A
0
5
10
15
20
25
30
Me
an
Cq
Va
lue
Gene RefSeq Accession Number
Comparison of Mean Cq Value
PrimeTime
Competitor A
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PrimeTime Assays excel with fast-cycling, Brilliant III
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Locating the pre-Designed qPCR page
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Search PrimeTime Pre-designed for RefSeq Number
Enter the RefSeq Number
Click “Submit”
www.idtdna.com/order/predesignedassay.aspx
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Step 5: Select the Same Exon Boundary
Select Exon Boundary
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IDT ZEN DOUBLE QUENCHED PROBES
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Requires overlap of donor emission spectra and acceptor absorption spectra andphysical proximity
Donor Acceptor Ro (Å)
Fluorescein Tamra 58
Fluorescein BHQ-1 60
Fluorescein BHQ-2 61
Fluorescein IBRQ 45
Fluorescein IBFQ 60
Fluorescein Dabcyl 46
Fluorescein Eclipse 60
Dual labeled probes: To capture fluorescence emitted by reporter dye
Proximity: Förster Radii
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5’-Fluorophore --------- 3’-Quencher
• Force reporter and quencher into proximity (Molecular Beacons, etc)
• Make probes shorter (even with LNA or MGB, it is hard to get probes much shorter than 14-18 bases)
5’-Fluorophore --------- Internal Quencher
• Place quencher internally on dT base (need a “T” nearby)
• Place quencher between bases (destabilizing Tm effect?)
Internal Quenching has many benefits, if the problem of destabilization can be solved
Methods to improve Quenching
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Modification DTm
ZEN + 3.8oC
BHQ2 - 0.6oC
Eclipse - 7.6oC
IBRQ - 8.6oC
C3 spacer - 8.7oC
S9 spacer - 19.4oC
All quenchers tested were destabilizing (lowered Tm) except ZEN
Insertion
Q
5’-ATCGTTGCTA-3’
3’-TAGCAACGAT-5’
^
Tm studies on Internal quenchers - insertion
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Actin qPCR Assay FAM-AGCTCACCATGGATGATGATATCGCC-Q
Lowest background was seen with the ZEN-DQ probe
Note the difference of Cq values
Zen-FQ DQP Probes vs. Standard design
RAW Baseline Adjusted
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RAW
Quencher placement makes a big difference
i8 – i10 best
i6 probably not being fully cleaved by Taq
Baseline Adjusted
Position Sequence HPRT qPCR assay
i12 FAM-ATGGTCAAGGT/ZEN/CGCAAGCTTGCTGGT-SpC3
i10 FAM-ATGGTCAAG/ZEN/GTCGCAAGCTTGCTGGT-SpC3
i8 FAM-ATGGTCA/ZEN/AGGTCGCAAGCTTGCTGGT-SpC3
i6 FAM-ATGGT/ZEN/CAAGGTCGCAAGCTTGCTGGT-SpC3
Optimization of Zen Assays
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5’-dye i10-Quencher 3’-Quencher
New DQP design gives lower baseline fluorescence while maintaining good signal strength
BHQ does not tolerate internal placement as well this way
RAW Baseline Adjusted
New Double Quencher Probe (DQP) design
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Using internal ZEN, the efficiency of quenching does not change as probes length increases
Zen-FQ design allows use of longer probes
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Summary for Double Quencher Probes
1. Proximity of Fluorophore and Quencher are crucial for efficient quenching in linear DLPs.
2. Most approaches to place a quencher internally within a sequence destabilize duplex formation (decrease Tm).
3. New ZEN Quencher stabilizes the duplex and increases Tm.
4. Novel internal ZEN + 3’-FQ (Double-Quencher format) gives excellent balance of low background combined with high signal generation and leads to increased sensitivity.
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IDT SciTools
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IDT SciTools – Where are they located?
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SciTools - OligoAnalyzer
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SciTool - PrimerQuest
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SciTools – RealTime PCR
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QUESTIONS?