1. science
2. biology
3. molecular biology

KiCqStart® Primers
Custom Primers
Black Hole Quencher®
Dual-Labeled Probes
Molecular Beacons
LightCycler® Probes
Scorpions® Probes
WellRED Primers
Primers and Fluorescent Probes
For Quantitative Real-Time PCR
and other Applications
bionucleics
JB_82324_Primers and Fluorescent Probes Brochure.indd 1
9/4/14 8:26 AM
bionucleics
Sigma® Life Science Custom Products
A Custom Solution for Every Project – Guaranteed
Predesigned and Custom Products
Sigma Life Science is recognized as the world’s leading supplier of custom oligonucleotides and peptide libraries for the global life science
research community. Originally founded in 1986 as Genosys Biotechnologies, Genosys was acquired by Sigma-Aldrich in 1998 to form
Sigma-Genosys. In 2005, Sigma-Aldrich acquired Proligo and its wide range of specialized DNA and siRNA products. Sigma-Genosys and
Sigma-Proligo harmonized product lines and continue to provide cutting-edge oligonucleotide technology, superior service and competitive
prices under the Sigma brand.
The Sigma Advantage
Global Manufacturing
We continuously invest in our worldwide operations and believe it
is not just the quality of our products that sets us apart but also the
quality of our service and technical expertise.
Sigma has oligonucleotide manufacturing sites around the globe,
in nine countries – Australia, Canada, Germany, India, Israel, Japan,
Singapore, UK and USA. Our global customers receive consistent
high-quality products.
The Sigma advantage includes:
•Our Commitment to Quality
•Outstanding Customer Service and Technical Support
•Global Manufacturing
Our Commitment to Quality
Quality is an integral part of our manufacturing process. Sigma
analyzes all oligonucleotides, including probes, by mass spectrometry,
ensuring the highest quality products. Complementary techniques,
such as analytical chromatography, are routinely used to verify
specifications are met. Our sizeable investment in state-of-the-art
analytical equipment provides industry-leading tools to develop and
monitor our process.
Our fluorescently-labeled probes are manufactured using a rigorous
process, including:
•Purification by reverse phase and/or ion exchange chromatography
•Electrospray mass spectral analysis
•Quality control documentation
•Amber packaging
Outstanding Customer Service and Technical Support
Our dedicated staff of highly-trained customer service specialists are
available via email or telephone to provide timely solutions to every
customer inquiry. Providing real-time status for orders, our customer
service teams demonstrate total commitment to customer satisfaction. With an extensive staff of Molecular Biologists and Chemists, our
technical experts are prepared to assist researchers with experimental
design, application support and troubleshooting. Whether contacting
us via the web, email or telephone, Sigma customers are provided
with best-in-class service and support.
JB_82324_Primers and Fluorescent Probes Brochure.indd 2
Manufacturing
Expertise of Our Scientists
With more than 25 years of experience in oligonucleotide synthesis,
we have the expertise to create the most technically challenging
custom biomolecules. Our scientists collaborate with researchers
around the world and together develop novel custom products. We
meet customer specifications, no matter how complex.
Quantitative PCR Tools
Sigma is pleased to offer a variety of educational tools for quantitative
PCR users including:
•Technical and troubleshooting guides
•PCR and qPCR technical manual
•Webinar series
•Workshops
9/4/14 8:26 AM
sigma.com/probes
3
The MIQE Guidelines
The potential applications for quantitative real-time PCR (qPCR)
have increased exponentially since the first description (Higuchi,
1993). However, researchers have been frustrated by complications
such as contamination, insufficient amplification, low sensitivity and
uncertainty about what constitutes a suitable statistical analysis. Until
recently, there has been a lack of consensus about how to handle
these obstacles.
An international research team published The MIQE (pronounced
Mykee) Guidelines in 2009 to address the challenges of performing
dependable qPCR measurements.
The MIQE Guidelines:
By Following the MIQE Guidelines You Will:
•Promote experimental transparency
•Ensure consistency between laboratories
•Maintain the integrity of the scientific literature
Publish or bring your product to market faster and with confidence
by adhering to The MIQE Guidelines.
To learn more about MIQE and download the paper, visit
sigma.com/miqe
Issues addressed by MIQE include–
•Experimental design
•Sample preparation
•Nucleic acid extraction
•Reverse transcription
•qPCR target information
•qPCR oligonucleotides
•qPCR protocol
•qPCR validation
•Data analysis
JB_82324_Primers and Fluorescent Probes Brochure.indd 3
9/4/14 8:26 AM
bionucleics
Detection Chemistries
Sigma offers primers, probes and reagents to support all qPCR assays.
DNA-Binding Dyes and Probes
Quantitative real-time PCR (qPCR) relies on real-time detection of
amplification products as they are formed in the reaction. This
can be accomplished using non-specific DNA binding dyes or
sequence-specific probes. These techniques and their benefits
are described below.
A Theoretical qPCR Assay
[F]
threshold
thr
Baseline
de
detection limit
There are several types of probe structures that can be used including:
•Dual-Labeled Probes
•Molecular Beacons
•LightCycler® Probes
•Scorpions® Probes
Each probe type enables researchers to measure an increase in
fluorescent signal that corresponds to an increase in the copy number
of the desired amplicon. In addition to the increase in sensitivity that
is gained from using sequence-specific probes for detection, these
probes can also be labeled with different fluorescent reporter dyes,
allowing detection of multiple targets within the same PCR reaction.
Benefits and Challenges of Multiplex Reactions
The use of probes labeled with different reporter dyes allows the
simultaneous detection and quantification of multiple target genes in
a single (multiplex) reaction.
Cq
Cq
Cq
Cq
Cycle #
Figure 1. During a qPCR assay, the progress of the reaction is monitored by tracking the increase
in fluorescence from an associated reporter dye molecule. The number of cycles required to
reach a threshold level of detection is the quantification cycle (Cq). The lower the Cq the higher
the initial concentration of target and vice versa.
Non-specific DNA Binding Dye Detection
SYBR® Green I binds non-specifically to double-stranded DNA.
Upon binding, the dye undergoes a conformational change resulting
in high fluorescent emission, allowing measurement of the total
amount of double-stranded product present in the reaction after
each amplification cycle.
SYBR Green I detection is a popular option due to its low cost and
ease of use. However, multiple double-stranded species that may be
present cannot be discriminated when using SYBR Green I. Within
any PCR, there is the potential for primer dimer formation and nonspecific amplification products. Therefore, melt curves must be used
after amplification for estimating the specificity of amplified products.
It may be difficult to obtain accurate quantification at low target
concentrations.
For this reason, many researchers use SYBR Green I detection for
initial screening, proof-of-concept experiments or common
applications, such as gene expression analysis, and progress to
probe-based detection for greater assay sensitivity and/or for
multiplex analysis.
Probe-based Detection
Probe-based detection methods rely on one or more fluorescently
labeled probes that are positioned between the two PCR primers.
Because the probe is sequence specific, it will only detect the
presence of a single amplicon within the reaction.
JB_82324_Primers and Fluorescent Probes Brochure.indd 4
There are situations in which multiplex reactions are beneficial
including:
•Limited Template Availability: The number of amplifications can
be maximized
•Large Numbers of Samples: Reducing the number of reactions
leads to cost savings
However, there are a variety of challenges when developing a multiplex assay including:
•Complex Design: The degree of difficulty increases with the
number of targets to be detected
•Optimization Reaction Conditions: All primer/probe sets need
similar reaction kinetics and the same buffer, therefore target
sensitivity may be reduced compared to similar singleplex reactions
Predesigned vs. Custom Assays
Predesigned assays offer convenience since no time or effort is
needed for primer and probe designs or deciding on oligonucleotide
specifications, such as the right manufacturing scale, purification,
yield, etc.
Sigma’s KiCqStart® Primers are ready-to-order (all specifications are
set), predesigned SYBR Green I Primers for RT-qPCR and are available
for a variety of species. They can be searched and ordered online at
sigma.com/ksprimers.
Sometimes, demanding assays require a greater level of attention
than can be found with predesigned assays. All Sigma’s detection
chemistries including SYBR Green I Primers, Dual-Labeled Probes,
Molecular Beacons, LightCycler Probes, and Scorpions Probes can be
customized to your exact specifications.
9/4/14 8:26 AM
sigma.com/probes
5
Assay Design
OligoArchitect™ Primer and Probe Design Solutions
OligoArchitect Consultative
Sigma® is pleased to offer OligoArchitect for all of your primer
and probe design requirements. OligoArchitect includes both
our complimentary online design tool and our unique
consultative service.
For more complex and demanding applications, utilize our
consultative service to ensure the success of your assay. With
personal consultation from our expert, technical support scientists,
your request, including all sequences and data analysis, will be MIQE
compliant and returned to you within 24-48 hours. If required, you will
also receive follow-up assay optimization, data analysis assistance and
troubleshooting support. Requests for consultative design service can
be submitted at sigma.com/probedesignonline.
OligoArchitect Online
For routine needs, improve your assay with our OligoArchitect online
design tool powered by the industry standard Beacon Designer™
(PREMIER Biosoft). The user-friendly interface utilizes the latest
algorithms, provides results in real time, supports templates up to
10,000 base pairs, and allows for the adjustment of input parameters
such as homopolymer run/repeat maximum length, G/C clamp length
and maximum primer pair TM mismatch.
Designs can be completed for traditional PCR or qPCR using the
following detection chemistries (Locked Nucleic Acid® (LNA®) can be
included in probe and beacon designs):
•SYBR® Green I Primers
•Dual-Labeled Probes
•Molecular Beacons
•LightCycler® Probes
•Scorpions® Probes
Our online design tool can be used for the following applications:
•Traditional PCR
•SNP detection
•Allele discrimination
•Pathogen detection
•Multiplexing
•Viral load quantification
•Gene expression analysis
•Gene copy determination
•Endpoint genotyping
All reported sequences, associated properties, and assay parameters
are available for export to Excel and convenient email ordering. Visit
sigma.com/probedesignonline to try OligoArchitect Online.
Designs can be completed for traditional PCR or qPCR using the
following detection chemistries (Locked Nucleic Acid (LNA) can be
included in probe and beacon designs):
•SYBR Green I Primers
•Dual-Labeled Probes
•Molecular Beacons
•LightCycler Probes
•Scorpions Probes
Designs can typically be completed for the following PCR and
qPCR applications:
•Traditional PCR
•SNP detection
•Allele discrimination
•Pathogen detection
•Multiplexing
•Viral load quantification
•Gene expression analysis
•Gene copy determination
•Endpoint genotyping
•Methylation analysis
•High-resolution melting analysis
•Transcript detection across
•
•
•
exon junctions
Haplotyping
Northern blotting
Southern blotting
Other applications:
FISH (fluorescence in situ hybridization)
•
Sigma’s consultative service uses Beacon Designer from PREMIER Biosoft
International. Blastn and mfold are used to determine assay specificity.
Bioinformatics Services
With recent growth in the volume and complexity of genomics and
proteomics research, it is necessary for scientists to be able to capture
and use this information. Sigma recognized this need and developed
state-of-the-art bioinformatics capabilities, including:
•A dedicated team of bioinformatics professionals with extensive
expertise in genomics and proteomics computing applications.
•An in-house system of hardware, software, tools and databases.
•
•
JB_82324_Primers and Fluorescent Probes Brochure.indd 5
Both proprietary and commercial software packages allow greater
flexibility in addressing researcher needs.
Microarray oligonucleotide design, AQUA™ Peptide design,
PEPscreen®: peptide library design, gene / transcript / proteomic
annotation, gene / protein function classification, microarray data
analysis and more.
Rapid and secure completion of projects. Entire genome
oligonucleotide microarray designs are completed in less than
two weeks. All sequence information is retained in a secure
environment using internal Blast databases for searches.
9/4/14 8:26 AM
bionucleics
Reaction Optimization
Proper optimization can increase the efficiency and sensitivity of a
qPCR assay.
Improved Assay Sensitivity and Specificity
Using Locked Nucleic Acid® (LNA®)
Primer and Probe Placement
LNA can be incorporated into any of our qPCR probe types, providing
enhanced sensitivity and specificity for your assay. LNA is a novel type
of nucleic acid analog containing a 2’-O, 4’-C methylene bridge. A
comparison of LNA to DNA is shown below:
In general, amplicons should be between 50-200 bases in length.
Shorter amplicons tend to be more tolerant of less than ideal reaction
conditions, improving the consistency of results.
When quantifying RNA targets, select primers spanning exon-exon
junctions to avoid amplification of contaminating genomic DNA in
cDNA samples.
Primer Optimization Improves Reaction Sensitivity
55
Fluorescence
50
45
40
Optimal
primer
concentration
15
Sub-optimal
primer
concentration
10
5
20
25
30
35
40
Cycle
Figure 2. Primer optimization assay using Sigma SYBR® Green mastermix. Optimization of primers
for the human UBC gene. All reactions contain exactly the same template but varying primer
concentrations. Assay highlights how variation in primer concentration has an impact on the
sensitivity of the assay. Platform: Rotor-Gene™ Corbett Research Ltd. Description of protocol
optimization is referenced: Nolan T, Hands RE, Bustin SA Quantification of mRNA using real-time
RT-PCR. Nature Protocols 2006; 1:1559-1582
Buffer Conditions
Assay performance may also benefit from optimization of buffer
components (particularly MgCl2) and the internal reference dye.
Optimizing the concentration of these components is especially
important when designing multiplex assays or singleplex assays in
which design of an appropriate probe/primer combination proves to
be difficult.
JB_82324_Primers and Fluorescent Probes Brochure.indd 6
DNAMonomer
Monomer
DNA
When used with any standard bases (A,C,G,T, or U), probes
synthesized using LNA have greater thermal stability than
conventional DNA or RNA and therefore form a stronger bond
with the complementary sequence.
Probe Sequence
20
15
LNAMonomer
Monomer
LNA
The introduction of LNA chemistry into a qPCR probe may result in
an increase in the TM of up to 8 °C per LNA monomer substitution
in medium salt conditions compared to a DNA fluorescent probe. It
is possible to optimize the TM level and the hybridization specificity
through specific placement of the LNA base(s) in the probe design as
shown below.
60
25
O
O
Optimization of primer and probe concentrations can improve the
detection level of a particular amplicon by approximately 10 cycles
depending on the sequence. Since a 3 Cq difference in amplicon
detection indicates approximately a 10 fold difference in template
concentration, this simple step can greatly improve both the accuracy
and sensitivity of the reaction.
30
Base
HO
O
Primer and Probe Concentration
35
Base
HO
LNA Bases
TM*
∆TM
∆TM/LNA
GTGATATGC
0
29 °C
––
––
GTGATATGC
3
44 °C
15 °C
+5 °C
GTGATATGC
9
64 °C
35 °C
+3.9 °C
* TM of duplex between probe and its complementary sequence
Note: The bolded and underlined bases denote LNA base
Incorporation of LNA into your qPCR probe can improve performance
of many assays, including:
•SNP Discrimination: The presence of a single base mismatch has
a greater destabilizing effect on the duplex formation between
a LNA fluorescent probe and its target nucleic acid than with a
conventional DNA fluorescent probe.
•Multiplex Assays: Incorporation of LNA bases allows simpler T
optimization, providing more flexibility in probe placement.
M
•Problematic Target Sequences: Shorter probes can be designed
to address traditionally problematic target sequences, such as
AT- or GC-rich regions, highly repetitive sequences or regions with
difficult secondary structure. Short regions of homology in aligned
sequences can also be targeted.
9/4/14 8:26 AM
sigma.com/probes
7
Data Analysis
As your qPCR experiments grow in complexity and the data volume
becomes overwhelming, rely on our expertise to help you advance
your research.
•Before beginning your experiment, we will evaluate and provide
recommendations for developing protocols.
into a comprehensive results report, including all statistical
analyses as well as publication-ready tables and graphs.
Design and Analysis Options:
2
MDH
M-Value (Anti Log2)
FBP
ADH2
1.6
1.4
1.2
ADH1
PGK
HSP
TPI
1
0.8
0.6
0.4
PDC
SUC
ADH4
IPPI
PDA
CYC
ADH6
ADH3
ADH5
MIG
•Purchase Sigma® qPCR probes
•Select desirable design and analysis options
•Send your experimental protocols and raw data in .xls or .mdf file
format to the email address below, and you will be contacted by
one of our expert biostatisticians
•After completing your experiment, we will process your raw data
1.8
Next Steps:
•Evaluation or development of assay design
•Identification of optimal reference genes
•Identification of marker gene combinations
•Quantification of unknown samples
•Determination of statistical significance
Benefits:
Bactin
0.2
0
Genes
Figure 3. Example reference gene selection that can be included with the results report.
Sigma’s Biostatistics service will allow you to maintain your focus on
critical data-gathering activities while avoiding the need to purchase
expensive software and train personnel in time-consuming statistical
analyses. You will obtain results promptly so that you can publish or
bring your product to market faster and with confidence.
To request service, send an email to [email protected]
JB_82324_Primers and Fluorescent Probes Brochure.indd 7
9/4/14 8:26 AM
bionucleics
KiCqStart® Primers
KiCqStart Primers are predesigned SYBR® Green I Primers for two-step
and one-step RT-qPCR. Available for a variety of species, the primer
pairs can be easily searched and ordered online.
Guaranteed Yields
Guaranteed
OD Yield
Appx. No.
of nmoles
Appx. No.
of μg
Appx. No.
of Reactions*
3
14
90
1,500
Choose KiCqStart Primers for:
•Gene expression analysis
•Validating MISSION® siRNA and shRNA knockdowns
*Estimate based on 14 nmoles or 30 µg for 3 OD and 450 nM in a 20 uL reaction. Estimate is
based on an average sequence length of 21 bases.
To learn more and order, visit
Benefits of Using KiCqStart Primers Include:
sigma.com/ksprimers
•Developed with sophisticated bioinformatics tools and validated in
Companion Products
silico to avoid off-target amplification and SNP loci (when possible)
•MIQE compliant – sequences are provided at the time of shipment
•Compatible with any thermal cycler
How KiCqStart Primers Work
KiCqStart Primers are PCR primers that work with SYBR Green I.
When free in solution with only ssDNA present, SYBR Green I emits
a low signal intensity. As the reaction progresses and the quantity
of dsDNA increases, more dye binds to the amplicons and hence,
signal intensity increases. The illustration below depicts
the mechanism.
Two-step RT-qPCR with KiCqStart Primers is easy with ReadyScript®
cDNA Synthesis Mix and KiCqStart SYBR Green qPCR ReadyMixes.
ReadyScript cDNA Synthesis Mix for Fast and Easy
Preparation of Your cDNA Templates for qPCR
ReadyScript cDNA Synthesis Mix is a sensitive and easy-to-use
solution for two-step RT-qPCR. This 5X concentrated mix provides
all necessary components (except RNA template) for first-strand
synthesis including: buffer, dNTPs, MgCl2, primers, RNase inhibitor
protein, ReadyScript reverse transcriptase and stabilizers. ReadyScript
is a RNase H(+) derivative of MMLV reverse transcriptase, optimized for
reliable cDNA synthesis over a wide dynamic range of input RNA.
Cat. No.
RDRT
1. Dye in solution emits
low fluorescence
2. Emission of the fluorescence by binding
SYBR Green I cycles between an unbound (denaturation) and
a bound (annealing through extension) state as the reaction
progresses, and signal intensity increases as the quantity of
amplicons increase in later cycles.
Product Features Include:
•Amount: 3 OD minimum yield
•Purification: RP cartridge
•Sequence Lengths: 18 – 24 bases
•Quality Control: 100% mass spectrometry
•Format: Supplied dry, two oligos, one per tube
JB_82324_Primers and Fluorescent Probes Brochure.indd 8
Product Description
ReadyScript cDNA Synthesis Mix
KiCqStart SYBR Green qPCR ReadyMix™ for All Real-Time
PCR Instruments
KiCqStart SYBR Green ReadyMixes for fast qPCR come in a ready-touse 2X formulation, which includes SYBR Green I dye, an antibodymediated Hot-Start Taq DNA polymerase, dNTPs, MgCl2, and
proprietary buffers and stabilizers. Just add primers and template, and
you will discover that ReadyMixes offer the following features and
benefits:
•Speed – assay results in as little as 33 minutes
•Quality – highly efficient and sensitive real-time PCR results
•Ease of use – little to no optimization required
Cat. No.
KCQS00
KCQS01
KCQS02
KCQS03
Product Description
KiCqStart SYBR Green qPCR ReadyMix
KiCqStart SYBR Green qPCR ReadyMix, Low ROX™
KiCqStart SYBR Green qPCR ReadyMix with ROX
KiCqStart SYBR Green qPCR ReadyMix, iQ™
9/4/14 8:26 AM
sigma.com/probes
9
Custom Primers
Forward and/or reverse primers are required for all custom probe
and beacon detection chemistries offered by Sigma.
Related Products
Choose Custom Primers for:
Easy Oligo:
•
•Molecular Beacons
•LightCycler® Probes
•Scorpions® Probes
•Amount: 3 OD
•Concentration: 100 µM
•Purification: Desalt
•Sequence Lengths: 15 – 30 bases
•Quality Control: 100% mass spectrometry
•Format: Supplied in solution
•Ships within 24 hours of order receipt
Dual-Labeled Probes
Benefits of Using Sigma’s Primers Include:
•Free design support with OligoArchitect
•Comprehensive technical support
•Quality guaranteed
Pure & Simple Primers:
Product Features Include:
•
•Purification: Desalt, Cartridge, HPLC, and PAGE
•Sequence Lengths: 10-120 bases (inquire for longer lengths)
•Modifications: Over 200 available, including dyes (fluorescent and
Scale: 0.025, 0.05, 0.2, 1.0, 10, and 15 µmole
non-fluorescent)
•Quality Control: 100% mass spectrometry
•Format: Supplied dry or in solution – tubes, plates, and mixed plates
(forward and reverse primers)
To learn more and order, visit
iScale™ Oligos
Guaranteed Yields
Appx. No.
Guaranteed OD Yield
3
5
12
40
400
600
•Amount: 3 OD
•Purification: Cartridge
•Sequence Lengths: 12 – 35 bases
•Quality Control: 100% mass spectrometry
•Format: Supplied dry
•Ships within 24 hours of order receipt
sigma.com/oligos
•WellRED Oligos are also available
Scale (µmole)
0.025
0.05
0.2
1.0
10.0
15.0
We offer a range of products when the focus is speed and simplicity.
Appx. No. of μg
96
160
384
1,280
12,800
19,200
of Reactions*
600
1,000
2,500
8,500
85,000
130,000
*Estimate based on 32 µg for 1 OD and 900 nM in a 25 µL reaction. Estimate is based on desalt
purification and an average sequence length of 21 bases.
•Larger quantities for high-throughput projects
•Quantity: 10, 25, 50, 100, 250, 500 mg (inquire for larger quantities)
•Purification: Desalt, RP-HPLC, IE-HPLC
•Sequence Lengths: 10 – 50 bases (inquire for longer lengths)
•Modifications: Over 200 available, including dyes (fluorescent and
non-fluorescent)
•Quality Control: 100% mass spectrometry
•Format: Supplied dry
To learn more and order, visit
sigma.com/iscaleoligos
JB_82324_Primers and Fluorescent Probes Brochure.indd 9
9/4/14 8:26 AM
bionucleics
Choosing the Right Custom Probe
There is a wide selection of possible custom probe types that can be used for qPCR, and each has advantages for different applications. The
summary below highlights the applications of commonly used custom probes (one red dot is good, two is better).
Application Reference Guide
SYBR® Green I
Primers
Dual-Labeled
Probes
••
••
•
•
•
Dual-Labeled
LNA® Probes
Molecular
Beacons*
LightCycler®
Probes*
Scorpions®
Probes*
••
••
••
••
••
••
••
•
•
•
••
•
••
•
••
••
•
•
•
•
•
••
•
••
••
••
••
••
••
••
••
APPLICATION
Mass screening
Microarray validation
Multiple target genes / few samples
SNP detection
Allele discrimination
Pathogen detection
•
Multiplexing
Viral load quantification
Gene expression analysis
•
Gene copy determination
•
••
•
••
•
Endpoint genotyping
In vitro quantification or detection
•
*LNA® can be incorporated into the probes for improved specificity.
Reporters, Quenchers and Instrument Compatibility
Dye Substitutes
Modern qPCR platforms typically have multiple detection channels
enabling flexibility in the choice of probe labels. It is important to
select the fluorescent labels which are compatible with the detection
channels for the qPCR instrument and to ensure the correct filter
settings or detection calibration for the instrument. The Reporters
and Instrument Compatibility table (see page 11) lists a selection of
some of the most widely used qPCR platforms and indicates which
fluorescent labels may be used. Please note that not all labels are
listed and many alternative reporters are available. For information on
the use of non-standard labels with these platforms, contact your local
technical service professional ([email protected]).
Several qPCR instruments utilize proprietary dyes which are not
generally available commercially, such as VIC™ and NED™. When
seeking dye alternatives, the following criteria are important:
•The excitation and detection wavelength are compatible with the
instrument light source and detection system
•For probes, the quencher effectively absorbs light at the emission
wavelength of the reporter
•The higher the extinction coefficient the brighter the dye, which
contributes to sensitive detection
•When using multiple dyes (multiplex) the excitation and emission
wavelengths of each dye must be independent to avoid cross talk
Quenchers
Quenching molecules are typically placed at the 3’ end of single
molecule probes such as Dual-Labeled Probes, Molecular Beacons
and Scorpions Probes. Quenchers may be fluorescent (TAMRA™) or
nonfluorescent molecules (DABCYL, Black Hole Quencher® (BHQ®).
For optimal performance, the quencher’s absorbance spectrum
should match the reporter’s emission spectrum as closely as possible.
Recommended reporter/quencher combinations can be found in the
Spectral Properties table on page 11. To learn more about Black Hole
Quencher, see page 12.
JB_82324_Primers and Fluorescent Probes Brochure.indd 10
9/4/14 8:26 AM
sigma.com/probes
11
Spectral Properties Table
Dye
Max.
EX (nm)
Max.
EM (nm)
6-FAM™
494
515
BHQ-1, TAMRA
Cyanine 3
JOE™
520
548
BHQ-1, TAMRA
Quasar® 570
TET™
521
536
BHQ-1, TAMRA
Cal Fluor® Gold 5401
522
541
BHQ-1
HEX™
535
555
BHQ-1, TAMRA
Cal Fluor Orange 5602
540
561
BHQ-1
TAMRA™
555
576
BHQ-2
2
JOE/TET alternative
2
VIC® alternative
1
Cyanine 3 alternative
4
TAMRA alternative
3
Compatible
Quencher
Dye
Max.
EX (nm)
Max.
EM (nm)
550
570
BHQ-2
548
566
BHQ-2
Cal Fluor Red 590
565
588
BHQ-2
ROX™
573
602
BHQ-2
Texas Red®
583
603
BHQ-2
Cyanine 5
651
674
BHQ-3
5
Quasar 670
647
667
BHQ-3
Cyanine 5.5
675
694
BHQ-3
3
4
Cyanine 5 alternative
5
Compatible
Quencher
Sigma® is a licensed supplier of a variety of dyes and quenchers and continually adds to its
portfolio of new chemistries. For assistance in the design of your probe and/or assays, visit
sigma.com/probedesignonline.
Reporters and Instrument Compatibility Table
Platform
SYBR®
Green I
FAM
HEX
JOE
ROX
TET
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Qiagen Rotor-Gene® 6000
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Eppendorf
Mastercycler® ep realplex
•
•
•
ABI 7900HT
ABI 7300
ABI 7500
ABI 7700
ABI 7000
ABI StepOne™
ABI StepOnePlus™
Bio-Rad iQ™ 5
Bio-Rad Opticon™ 2
Bio-Rad Chromo4™
Bio-Rad MyiQ™
Bio-Rad MiniOpticon™
Bio-Rad CFX96™
BIo-Rad SFX384™
Agilent Mx4000®
Agilent Mx3000P®
Agilent Mx3005P®
Roche LightCycler®
Roche LightCycler 2
Roche LightCycler 480
Cepheid SmartCycler®
Cepheid SmartCycler II
Cyanine
3
Cyanine TAMRA Texas Red LC Red 640
5
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
LC Red
705
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Note: Not all qPCR instruments or reporters are listed.
Contact the instrument manufacturer for details on compatible reporters.
JB_82324_Primers and Fluorescent Probes Brochure.indd 11
9/4/14 8:26 AM
Abs (D
RFU (JO
6-FAM
bionucleics
250
300
350
400
450 500
550
600
650
700
750
Wavelength (nm)
Black Hole Quencher®
T9
Abs
Two commonly used quenchers, TAMRA™ and DABYCL, limit the
ultimate sensitivity and flexibility of qPCR. TAMRA is not a dark
quencher and therefore contributes to an overall increase in
background because of its own native fluorescence. DABCYL, though
a dark quencher, has an inadequate absorption footprint that overlaps
very poorly with reporter dyes emitting above 480 nm (Figure 4).
BHQ-3 (672 nm)
BHQ-2 (579 nm)
BHQ-1 (534 nm)
6-FAM (518)
TET (538)
HEX (553)/JOE (554)
Cyanine 3 (565)
TAMRA (583)
ROX (607)
LC Red (640)
Cyanine 5
(667)
Abs (DABCYL-T9)
RFU (JOE, TET, FAM)
DABCYL-T9
T9
JOE
TET
6-FAM
250
300
350
400
450 500
550
600
650
700
750
Wavelength (nm)
Figure 5. Absorption spectra of the three BHQ variants (conjugated to T-9 and normalized to the
poly-T absorbance of 260 nm) with the emission maxima of the commonly-used reporters 6-FAM,
TET, HEX™, Cyanine 3, TAMRA, ROX™, LC Red 640, and Cyanine 5.
250
300
350
400
450 500
550
600
650
700
In addition to better spectral overlap, BHQ probes have much higher
signal-to-noise ratios when compared to the corresponding DABCYL
or TAMRA probes (Figure 6).
750
Wavelength (nm)
5’-FAM Probes
Figure 4. Absorption spectra of DABCYL-T9 with the emission spectra of the commonly-used
reporters 6-FAM™, TET™, and JOE™. DABCYL was normalized at the poly-T absorbance (260 nm).
RFU = relative fluorescence unit.
5’-Cyanine 3 Probes
9.0
80
7.5
60
6-FAM (518)
Though DABYCL works well in MolecularTET
Beacons
(structured probes
(538)
HEX (553)/JOE
(554)
with paired reporter and
static quenching),
T9quencher that utilize
3 (565)
its absorption maximum of 474 nm places itCyanine
below
the
maxima of
TAMRA (583)
the reporters shown in Figure 4, therefore it is aROX
poor
choice for
(607)
Red (640) and
Dual-Labeled Probes (linear probes with separatedLCreporter
Cyanine 5
BHQ-3
(672
nm)
quencher that utilize FRET quenching).
(667)
4.5
S:N
S:N
6.0
20
Abs
1.5
0.0
DAB
BHQ-1
BHQ-2
DAB
3’ Quencher
5’-Cyanine 3 Probes
BHQ-1
3’ Quencher
5’-Cyanine 5 Probes
80
120
60
90
40
60
S:N
S:N
S:N
0
TAM
BHQ-2 (579 nm)
(534 nm)
Black Hole QuencherBHQ-1
1 (BHQ®-1)
(with an absorption maximum of 534
nm) is absorbed at higher wavelengths than DABCYL and is directly
5’-FAM
superimposable with the emission maxima of FAM, TET
andProbes
JOE. This
provides a significant increase in both static
and FRET quenching
9.0
efficiencies, which makes it an excellent 7.5
quencher for Molecular
350 400 450 500 550 600 650 700 750
Beacons250
and 300
Dual-Labeled
Probes. As shown in Figure 5, BHQ-1, 2, and
6.0
Wavelength
(nm)
3 cover the spectrum from 480
nm into the
near IR making it possible
to utilize reporters that emit anywhere in4.5this range.
40
3.0
3.0
20
30
1.5
0.0
0
TAM
DAB
BHQ-1
3’ Quencher
BHQ-2
0
DAB
BHQ-1
3’ Quencher
BHQ-2
DAB
BHQ-2
BHQ-3
3’ Quencher
Figure 6. Signal-to-noise (S:N) ratios were calculated by dividing the fluorescence signal of a
25mer in the presence of a five-fold excess of an exact complementary target sequence by the
fluorescence intensity of the probe alone.
JB_82324_Primers and Fluorescent Probes Brochure.indd 12
9/4/14 8:26 AM
BHQ-2
sigma.com/probes
Finally, as shown in Figure 7, the BHQ variants readily permit singletube multiplexing due to the increased variety of reporters that can
be effectively quenched with little or no cross-talk. This simplifies the
design, implementation, and interpretation of multiplexed assays.
13
In Summary, Black Hole Quencher:
1) has no native fluorescence (emits heat instead of light), resulting
in lower background fluorescence
2) has increased signal-to-noise ratio, providing higher sensitivity
3) enables a wider choice of reporters for multiplexing
Normalized Emission
6-FAM
TET
JOE
450
500
550
600
650
700
750
Wavelength (nm)
Normalized Emission
6-FAM
TAM
ROX
CYANINE 5
450
500
550
600
650
700
750
Wavelength (nm)
Figure 7. The unique characteristics of the BHQ variants permit flexibility in the choice of
spectrally well-resolved reporters, which enable single-tube multiplexing with little or no
cross-talk.
JB_82324_Primers and Fluorescent Probes Brochure.indd 13
9/4/14 8:26 AM
bionucleics
Dual-Labeled Probes
Dual-Labeled Probes are the most common probe type for qPCR and
are often referred to as hydrolysis probes.
Choose Dual-Labeled Probes for:
•Microarray validation
•Multiple target genes /
•Multiplexing
•Viral load quantification
•Gene expression analysis
•Gene copy determinations
few samples
•Pathogen detection
Perfect for validating MISSION® siRNA and shRNA knockdowns
Product Features Include:
•Amounts: 1, 3, 5, and 10 OD
•Purification: HPLC
•Sequence Lengths: 15 - 40 bases
•Quality Control: 100% mass spectrometry
•Format: Supplied dry in amber tubes
•Custom formats available (normalization, special plates, etc.)
Sigma’s probes are provided in a format to simplify your experimental
planning.
Guaranteed Yields
Benefits of Using Dual-Labeled Probes Include:
•Design simplicity for sequence specificity
•Extensive availability of reporter/quencher combinations
•Increased sensitivity
Guaranteed
OD Yield
Appx. No.
of nmoles
Appx. No.
of μg
Appx. No.
of Reactions*
1
4
32
800
3
12
96
2,400
5
20
160
4,000
10
40
320
8,000
Add LNA® to Your Probe for:
•Increased thermal stability and hybridization specificity
•Greater accuracy in SNP detection, allele discrimination, and in vitro
quantification or detection
* Estimate is based on 4 nmoles or 32 μg for 1 OD and 200 nM in a 25 µL reaction
(5.0 pmol/reaction). Estimate is based on an average sequence length of 25 bases.
target sequences
The most common reporter and quencher combinations are
listed below:
•Easier and more sensitive probe designs for problematic
Spectral Properties Table
How Dual-Labeled Probes Work
A Dual-Labeled Probe is a single-stranded oligonucleotide
labeled with two different dyes. A reporter dye is located at
the 5’ end and a quencher molecule located at the 3’ end. The
quencher molecule inhibits the natural fluorescence emission of
the reporter by fluorescence resonance energy transfer (FRET).
The illustration below depicts the mechanism.
R
Q
3’ Quencher
Taq
DNA
polymerase
Q
2. Emission of the fluorescence
by hydrolysis
The primer is elongated by the polymerase and the probe binds
to the specific DNA template. Hydrolysis releases the reporter
from the probe/target hybrid, causing an increase in fluorescence.
The measured fluorescence signal is directly proportional to the
amount of target DNA.
JB_82324_Primers and Fluorescent Probes Brochure.indd 14
6-FAM™
494
515
BHQ®-1, TAMRA™
JOE™
520
548
BHQ-1, TAMRA
TET™
521
536
BHQ-1, TAMRA
Cal Fluor® Gold 540
522
541
BHQ-1
HEX™2
535
555
BHQ-1, TAMRA
Cal Fluor Orange 560
540
561
BHQ-1
TAMRA™
555
576
BHQ-2
Cyanine 3
550
570
BHQ-2
Quasar® 570
548
566
BHQ-2
Cal Fluor Red 5904
565
588
BHQ-2
ROX™
573
602
BHQ-2
Texas Red®
583
603
BHQ-2
Cyanine 5
651
674
BHQ-3
Quasar 6705
647
667
BHQ-3
694
BHQ-3
3
Amplified Target DNA
1. Probe in solution emits
low fluorescence
Max. EM Compatible
(nm)
Quencher
2
R
3’ Quencher
Max. EX
(nm)
1
5’ Reporter
5’ Reporter
Dye
Cyanine 5.5
JOE/TET alternative
2
VIC® alternative
1
675
Cyanine 3 alternative
4
TAMRA alternative
3
Cyanine 5 alternative
5
Sigma® is a licensed supplier of a variety of dyes and quenchers and
continually adds to its portfolio of new chemistries. For assistance in
the design of your Dual-Labeled Probe assays, use our online design
tool at sigma.com/probedesignonline.
9/4/14 8:26 AM
sigma.com/probes
15
Molecular Beacons
Molecular Beacons are structured probes that are highly sensitive,
sequence specific, and are used for sequence detection in qPCR and
in vitro studies.
Choose Molecular Beacons for:
•SNP detection
•Allele discrimination
•Pathogen detection
•Multiplexing
•Viral load quantification
•Gene expression analysis
•Gene copy determination
•End point genotyping
•In vitro quantification or
detection
Benefits for Using Molecular Beacons Include:
Product Features Include:
•Amounts: 1, 3, 5, and 10 OD
•Purification: HPLC
•Sequence Lengths: 15 - 40 bases
•Quality Control: 100% mass spectrometry
•Format: Supplied dry in amber tubes
•Custom formats available (normalization, special plates, etc.)
Sigma’s probes are provided in a format to simplify your experimental
planning.
Guaranteed Yields
•Probe preserved during the reaction
•Increased specificity
Guaranteed
OD Yield
Appx. No.
of nmoles
Appx. No.
of μg
Appx. No.
of Reactions*
Add LNA to Your Probe for:
1
3
32
600
•Increased thermal stability and hybridization specificity
•Greater accuracy in SNP detection, allele discrimination, and in vitro
3
9
96
1,800
5
15
160
3,000
10
30
320
6,000
quantification or detection
•Easier and more sensitive probe designs for problematic target
sequences
The most common reporter and quencher combinations are
listed below:
How Molecular Beacons Work
A Molecular Beacon is a single-stranded bi-labeled fluorescent
probe held in a hairpin-loop conformation (around 20 to 25 nt) by
complementary stem sequences (around 4 to 6 nt) at both ends
of the probe. The 5’ and 3’ ends of the probe contain a reporter
and a quencher molecule, respectively. The loop is a singlestranded DNA sequence complementary to the target sequence.
The close proximity of the reporter and quencher causes the
quenching of the natural fluorescence emission of the reporter.
The structure and mechanism of a Molecular Beacon is shown
below.
Loop
Sequence
Loop Sequence
5’ Reporter
3’ Quencher
Stem
Sequence
Q
3’ Quencher
R
R
5’ Reporter
1. Unbound beacon with
quenched fluorescence
* Estimate is based on 3 nmoles or 32 μg for 1 OD and 200 nM in a 25 µL reaction (5.0 pmol/
reaction). Estimate is based on an average sequence length of 30 bases.
Q
Amplified Target DNA
2. Bound beacon with
unquenched fluorescence
Molecular Beacons hybridize to their specific target sequence
causing the hairpin-loop structure to open and separate the 5’
end reporter from the 3’ end quencher. As the quencher is no
longer in proximity to the reporter, fluorescence emission takes
place. The measured fluorescence signal is directly proportional
to the amount of target DNA.
JB_82324_Primers and Fluorescent Probes Brochure.indd 15
Dye
Max. EX
(nm)
Max. EM Compatible
(nm)
Quencher
6-FAM™
494
515
BHQ-1, DABCYL
Fluorescein
495
520
BHQ-1, DABCYL
JOE™
520
548
BHQ-1, DABCYL
TET
521
536
BHQ-1, DABCYL
HEX
535
555
BHQ-1, DABCYL
Cyanine 3
550
570
BHQ-2, DABCYL,
ROX™
573
602
BHQ-2, DABCYL
Texas Red®
583
603
BHQ-2, DABCYL,
Cyanine 5
651
674
BHQ-3, DABCYL,
Cyanine 5.5
675
694
BHQ-3, DABCYL,
Sigma® is a licensed supplier of a variety of dyes and quenchers and
continually adds to its portfolio of new chemistries. For assistance in
the design of your Molecular Beacon assays, use our online design
tool at sigma.com/probedesignonline.
9/4/14 8:26 AM
bionucleics
LightCycler® Probes
LightCycler probes are highly sensitive and sequence-specific
fluorescent probes designed for use with the Roche LightCycler
instruments.
Choose LightCycler Probes for:
•
•
•
•
•
•
•
SNP detection
Allele discrimination
Pathogen detection
Multiplexing
Add LNA® to Your Probe for:
•Increased thermal stability and hybridization specificity
•Greater accuracy in SNP detection and allele discrimination
•Easier and more sensitive probe designs for problematic
target sequences
Viral load quantification
Gene expression analysis
Gene copy determination
Several reporters are available and are suitable for multiplex analysis.
Benefit of Using LightCycler Probes include:
•Increased specificity
•Probe preserved during the reaction
•Increased thermal stability and hybridization specificity
•Greater accuracy in SNP detection and allele discrimination
•Easier and more sensitive probe designs for problematic
Product Features Include:
•Amounts: 0.1, 0.25, 1.5, and 15 OD
•Purification: HPLC
•Sequence Lengths: 15 - 40 bases
•Quality Control: 100% mass spectrometry
•Format: Supplied dry in amber tubes
•Custom formats available (normalization, special plates, etc.)
Sigma’s probes are provided in a format to simplify your
experimental planning.
Guaranteed Yields of LightCycler Probes
target sequences
Guaranteed
OD Yield
Appx. No.
of nmoles
Appx. No.
of μg
0.1
0.4
3.2
80
0.25
1
8
200
How LightCycler Probes Work
A LightCycler Probe system consists of a pair of single-stranded
fluorescent-labeled oligonucleotides. Oligo Probe 1 is labeled at
the 3’ end with a donor reporter and Oligo Probe 2 is labeled at
its 5’ end with one of a few available acceptor reporters. The free
3’ hydroxyl group of Probe 2 must be blocked with a phosphate
group (P) to prevent DNA polymerase extension. There should
be a space of 1 to 5 nt to separate the two probes from each
other. The structures and mechanism of a LightCycler probe are
shown below.
3’ Donor Fluorophore (FD)
FD
Oligo Probe 1
FD
5’ Acceptor
Fluorophore (FA)
FA
FA
P
FRET
P
Amplified Target DNA
Oligo Probe 2
1. Probes in solution emit
low fluorescence
2. Emission through fluorescence
resonance energy transfer
During the annealing step of qPCR, the PCR primers and the
LightCycler Probes hybridize to their specific target regions
bringing the donor dye into close proximity to the acceptor dye.
When the donor dye is excited by light from the LightCycler
instrument, energy is transferred by FRET from the donor to
the acceptor dye. The acceptor reporter’s emission wavelength
is detected. The increase in fluorescence signal is directly
proportional to the amount of target DNA.
JB_82324_Primers and Fluorescent Probes Brochure.indd 16
Appx. No.
of Reactions*
1.5
6
48
1,200
15
60
480
12,000
* Estimate is based on 4 nmoles or 32 μg for 1 OD and 200 nM in a 25 µL reaction (5.0 pmol/
reaction). Estimate is based on an average sequence length of 25 bases.
The recommended constructs for Oligo Probe 1 and Oligo Probe 2 are
listed in the tables below:
Labels and Modifications for LightCycler Probes
Oligo Probe 1
3’ end
Fluorescein
Oligo Probe 2*
3’ end
Phosphate
5’ end
LightCycler Red 610, 640, 670 and 705
*For enhanced discrimination, LNA can be incorporated into Probe 2
Sigma® is a licensed supplier of a variety of dyes and quenchers and
continually adds to its portfolio of new chemistries. For assistance in
the design of your LightCycler Probe assays, use our online design tool
at sigma.com/probedesignonline.
9/4/14 8:26 AM
sigma.com/probes
17
Scorpions® Probes
Scorpions probes are highly sensitive, sequence specific, bi-labeled
fluorescent probe/primer hybrids designed for qPCR.
Choose Scorpions for:
•SNP detection
•Allele discrimination
•Pathogen detection
•Multiplexing
•Viral load quantification
•Gene expression analysis
•Gene copy determination
•Endpoint genotyping
Because the probe and primer are incorporated into a single
molecule, the reaction kinetics of this probe are extremely fast. The
reaction leading to generation of a fluorescent signal is essentially
instantaneous and occurs prior to any competing side reactions. This
enables Scorpions Probes to provide stronger signals, shorter reaction
times and better discrimination than other conventional bi-molecular
mechanisms. It also allows for more reliable probe design.
Benefits of Using Scorpions Probes Include:
•Increased specificity
•Fast amplicon detection
•Exceptional signal-to-noise (bi-probes typically yield a stronger
Add LNA to Your Probe for:
•Increased thermal stability and hybridization specificity
•Greater accuracy in SNP detection and allele discrimination
•Easier and more sensitive probe designs for problematic
target sequences
Product Features Include:
•Amounts: 1, 5, and 10 OD
•Purification: HPLC
•Sequence Lengths: 30 - 60 bases (Uni-Probe) and 15 - 45 bases
(Bi-Probe)
•Quality Control: 100% mass spectrometry
•Format: Supplied dry in amber tubes
•Custom formats available (normalization, special plates, etc.)
Sigma’s probes are provided in a format to simplify your experimental
planning.
Guaranteed Yields
Guaranteed
OD Yield
Appx. No.
of nmoles
Appx. No.
of μg
Appx. No.
of Reactions*
signal when compared to uni-probes)
1
2
32
400
5
10
160
2,000
How Uni-Probe Scorpions Probes Work
10
20
320
4,000
The Scorpions Uni-Probe consists of a single-stranded bi-labeled
fluorescent probe sequence held in a hairpin-loop conformation
with a 5’ end reporter and an internal quencher directly linked to
the 5’ end of a PCR primer via a blocker. The blocker prevents the
polymerase from extending the PCR primer.
Blocker
Loop
Sequence
Internal
Quencher
Stem
Sequence
Q
5’ Reporter
Blocker
5’ Reporter
Q
Internal
Quencher
R
Loop
Sequence
PCR Primer
Target DNA
1. Quenching of the fluorescence
PCR Primer
R
Newly Synthesized
DNA Strand
Complementary Sequence
2. Emission of the fluorescence
At the beginning of the qPCR, the polymerase extends the PCR
primer and synthesizes the complementary strand of the specific
target sequence. During the next cycle, the hairpin-loop unfolds
and the loop-region of the probe hybridizes intramolecularly to
the newly synthesized target sequence. Now that the reporter
is no longer in close proximity to the quencher, fluorescence
emission may take place. The fluorescent signal is detected by the
qPCR instrument and is directly proportional to the amount of
target DNA.
To learn about the bi-probe mechanism, please visit sigma.com/
probes, and click Learn More under Scorpions Probes.
JB_82324_Primers and Fluorescent Probes Brochure.indd 17
* Estimate is based on 2 nmoles or 32 μg for 1 OD and 200 nM in a 25 µL reaction (5.0 pmol/
reaction). Estimate is based on an average sequence length of 50 bases (Uni-Probe).
The available reporter and quencher combinations are listed below.
Scorpions Probes include a HEG (hexathylene glycol) blocker.
Uni-Probe
5’ Reporter
Internal Quencher
Fluorescein, 6-FAM™, HEX™, TET™, TAMRA™,
JOE™, ROX™, Cyanine 3, Cyanine 5, Cyanine 5.5,
Texas Red®, Rhodamine, Rhodamine Green™,
Rhodamine Red™, Oregon Green® 488, Oregon
Green 500, Oregon Green 514
DABCYL dT,
BHQ®-1,
BHQ-2,
BHQ-3
Bi-Probe
5’ Reporter
3’ Quencher
Fluorescein, 6-FAM, HEX, TET, TAMRA, JOE, ROX,
Cyanine 3, Cyanine 5, Cyanine 5.5, Texas Red,
Rhodamine, Rhodamine Green, Rhodamine
Red, Oregon Green 488, Oregon Green 500,
Oregon Green 514
TAMRA, DABCYL,
BHQ-1,
BHQ-2,
BHQ-3
Sigma® is a licensed supplier of a variety of dyes and quenchers and
continually adds to its portfolio of new chemistries. For assistance in
the design of your Scorpions Probe assays, use our online design tool
at sigma.com/probedesignonline.
9/4/14 8:26 AM
bionucleics
WellRED Primers
Sigma is licensed by Beckman Coulter, Inc., to supply WellRED
dye-labeled oligos designed for use with the CEQ™/GeXP Genetic
Analysis Systems and may be used for direct hybridization or in PCR
amplification. WellRED dye-labeled oligos are DNA oligonucleotides
labeled with WellRED dyes, D2, D3 or D4, at the 5’ end.
WellRED Specifications
Yields
Purification
Length
Backbone
Format
Modifications
Packaging
Quality Controls
Technical Datasheet &
Labels
Additional Services
Shipping Schedule
Cartridge: 2 and 4 OD
HPLC: 5, 10, 50 and 100 OD
PAGE: 1 OD
Cartridge, HPLC, PAGE
7 – 100 bases
Phosphodiester
Supplied Dry
D2-PA, D3-PA or D4-PA at the 5’ end
2 mL Opaque Tubes
MALDI-TOF MS, Electrospray Ionization MS,
OD by UV Spectroscopy
Includes yield, Tm, MW, and µg/OD
Duplicate set of labels with each order
Aliquoting into tubes
MSQC traces available upon request
Shipped within 4 to 5 business days
Yields and Estimated Number of Assays
Yield (OD)
Purification
Approx. yield
(nmol*)
Estimated assays
(reactions)
1
PAGE
5
2
4
5
Cartridge Cartridge HPLC
10
20
25
500-1,000 1,0002,000
2,0004,000
2,5005,000
10
HPLC
50
5,00010,000
*Estimate 1 OD = 5 nmol = 30 µg, for a 20 base oligo
Spectral Properties of WellRED Dyes
WellRED Oligo
D2-PA
D3-PA
D4-PA
Excitation
maximum (nm)
750
685
650
Emission
maximum (nm)
770
706
670
Molar extinction
coefficient
170,000
224,000
203,000
To learn more and order, visit
sigma.com/wellred
Benefits of WellRED Dye Chemistry
•WellRED oligos use cyanine-based fluorescent dyes with high
extinction coefficients that absorb in the near-infrared region,
thereby reducing background noise from biological materials,
resulting in highly sensitive detection.
•The CEQ/GeXP Genetic Analysis Systems enable DNA samples to be
kept in linear form during analysis. The CEQ/GeXP Genetic Analysis
Systems’ online denaturation process prevents any reformation
of secondary structures, especially during a long run without any
manual steps.
•WellRED dye chemistry and pre-heating enable the CEQ/GeXP
Genetic Analysis Systems to resolve complex structures, where other
systems fail.
JB_82324_Primers and Fluorescent Probes Brochure.indd 18
9/4/14 8:26 AM
sigma.com/probes
19
OligoEvaluator™
Sigma is pleased to offer OligoEvaluator, our online oligonucleotide
sequence calculator.
OligoEvaluator™
Functionality
Module
Analysis
Resuspension
Dilution
Features
Enter up to 10 sequences at a time, and the tool
returns values for all major physical properties, such
as molecular weight, melting temperature, secondary
structure, and primer dimer formation (secondary
structure and primer dimer formation information
provided in simple-to-interpret text format, e.g.
secondary structure--strong)
Enter the quantity, desired concentration, and
molecular weight, and the tool returns the volume
of water or buffer needed to resuspend a dry
oligonucleotide
Enter the stock (resuspension) concentration, desired
final volume, and desired final concentration, and
the tool returns the volume of stock solution needed
for dilution
Advanced Features
•Review secondary structure analysis and primer dimer formation
OligoEvaluator Input Inferface
to check for problematic folding
•Check for oligonucleotide sequence homology with BLAST
Algorithm
•Powered by PREMIER Biosoft
JB_82324_Primers and Fluorescent Probes Brochure.indd 19
All reported properties are available for export to a convenient Excel
template. Your OligoArchitect™ login can be used for OligoEvaluator.
To try OligoEvaluator, visit sigma.com/oligocalculator.
9/4/14 8:26 AM
Sigma-Aldrich ® Worldwide Offices
Argentina
Free Tel: 0810 888 7446
Tel: (+54) 11 4556 1472
Fax: (+54) 11 4552 1698
Australia
Free Tel: 1800 800 097
Free Fax: 1800 800 096
Tel: (+61) 2 9841 0555
Fax: (+61) 2 9841 0500
Austria
Tel: (+43) 1 605 81 10
Fax: (+43) 1 605 81 20
Belgium
Free Tel: 0800 14747
Free Fax: 0800 14745
Tel: (+32) 3 899 13 01
Fax: (+32) 3 899 13 11
Brazil
Free Tel: 0800 701 7425
Tel: (+55) 11 3732 3100
Fax: (+55) 11 5522 9895
Canada
Free Tel: 1800 565 1400
Free Fax: 1800 265 3858
Tel: (+1) 905 829 9500
Fax: (+1) 905 829 9292
Chile
Tel: (+56) 2 495 7395
Fax: (+56) 2 495 7396
China
Free Tel: 800 819 3336
Tel: (+86) 21 6141 5566
Fax: (+86) 21 6141 5567
Czech Republic
Tel: (+420) 246 003 200
Fax: (+420) 246 003 291
France
Free Tel: 0800 211 408
Free Fax: 0800 031 052
Tel: (+33) 474 82 28 88
Fax: (+33) 474 95 68 08
Korea
Free Tel: (+82) 80 023 7111
Free Fax: (+82) 80 023 8111
Tel: (+82) 31 329 9000
Fax: (+82) 31 329 9090
Germany
Free Tel: 0800 51 55 000
Free Fax: 0800 64 90 000
Tel: (+49) 89 6513 0
Fax: (+49) 89 6513 1160
Malaysia
Tel: (+60) 3 5635 3321
Fax: (+60) 3 5635 4116
Hungary
Ingyenes telefonszám: 06 80 355 355
Ingyenes fax szám: 06 80 344 344
Tel: (+36) 1 235 9063
Fax: (+36) 1 269 6470
India
Telephone
Bangalore: (+91) 80 6621 9400
New Delhi: (+91) 11 4358 8000
Mumbai: (+91) 22 2570 2364
Hyderabad: (+91) 40 4015 5488
Kolkata: (+91) 33 4013 8003
Fax
Bangalore: (+91) 80 6621 9550
New Delhi: (+91) 11 4358 8001
Mumbai: (+91) 22 4087 2364
Hyderabad: (+91) 40 4015 5488
Kolkata: (+91) 33 4013 8000
Ireland
Free Tel: 1800 200 888
Free Fax: 1800 600 222
Tel: (+353) 402 20370
Fax: (+ 353) 402 20375
Israel
Free Tel: 1 800 70 2222
Tel: (+972) 8 948 4100
Fax: (+972) 8 948 4200
Mexico
Free Tel: 01 800 007 5300
Free Fax: 01 800 712 9920
Tel: (+52) 722 276 1600
Fax: (+52) 722 276 1601
The Netherlands
Free Tel: 0800 022 9088
Free Fax: 0800 022 9089
Tel: (+31) 78 620 5411
Fax: (+31) 78 620 5421
New Zealand
Free Tel: 0800 936 666
Free Fax: 0800 937 777
Tel: (+61) 2 9841 0555
Fax: (+61) 2 9841 0500
Norway
Tel: (+47) 23 17 60 00
Fax: (+47) 23 17 60 10
Poland
Tel: (+48) 61 829 01 00
Fax: (+48) 61 829 01 20
Portugal
Free Tel: 800 202 180
Free Fax: 800 202 178
Tel: (+351) 21 924 2555
Fax: (+351) 21 924 2610
Denmark
Tel: (+45) 43 56 59 00
Fax: (+45) 43 56 59 05
Italy
Free Tel: 800 827 018
Tel: (+39) 02 3341 7310
Fax: (+39) 02 3801 0737
Russia
Tel: (+7) 495 621 5828
Fax: (+7) 495 621 6037
Finland
Tel: (+358) 9 350 9250
Fax: (+358) 9 350 92555
Japan
Tel: (+81) 3 5796 7300
Fax: (+81) 3 5796 7315
Singapore
Tel: (+65) 6779 1200
Fax: (+65) 6779 1822
Enabling Science to
Improve the Quality of Life
Order/Customer Service (800) 234-5362 • Fax (281) 363-5909
Technical Service (800) 325-5832 • sigma-aldrich.com/techservice
Development/Custom Manufacturing Inquiries
(800) 244-1173
Safety-related Information sigma-aldrich.com/safetycenter
Slovakia
Tel: (+421) 255 571 562
Fax: (+421) 255 571 564
South Africa
Free Tel: 0800 1100 75
Free Fax: 0800 1100 79
Tel: (+27) 11 979 1188
Fax: (+27) 11 979 1119
Spain
Free Tel: 900 101 376
Free Fax: 900 102 028
Tel: (+34) 91 661 99 77
Fax: (+34) 91 661 96 42
Sweden
Tel: (+46) 8 742 4200
Fax: (+46) 8 742 4243
Switzerland
Free Tel: 0800 80 00 80
Free Fax: 0800 80 00 81
Tel: (+41) 81 755 2828
Fax: (+41) 81 755 2815
United Kingdom
Free Tel: 0800 717 181
Free Fax: 0800 378 785
Tel: (+44) 1747 833 000
Fax: (+44) 1747 833 313
United States
Toll-Free: 800 325 3010
Toll-Free Fax: 800 325 5052
Tel: (+1) 314 771 5765
Fax: (+1) 314 771 5757
Vietnam
Tel: (+84) 3516 2810
Fax: (+84) 6258 4238
Internet
sigma-aldrich.com
World Headquarters
3050 Spruce St.
St. Louis, MO 63103
(314) 771-5765
sigma-aldrich.com
©2014 Sigma-Aldrich Co. LLC. All rights reserved. SIGMA and SIGMA-ALDRICH are trademarks of Sigma-Aldrich Co. LLC, registered in the US and other countries. Sigma brand products are sold through Sigma-Aldrich, Inc. Purchaser must determine the
suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see product information on the Sigma-Aldrich website at www.sigmaaldrich.com and/or on the reverse side of the invoice or packing slip. Where bio
begins, JumpStart and ReadyMix are trademarks of Sigma-Aldrich Co. LLC. PEPscreen® is a registered trademark of Sigma-Aldrich Co. LLC. KiCqStart is a registered trademark used under license. 5-FAM™, 6-FAM™ JOE™, TET™, HEX™, ROX™, TAMRA™, and VIC®
and are trademarks and registered trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries. Inc. Molecular Beacons are sold under license from PHRI, City of New York, Inc. LightCycler® Probes sold under license from Roche
Diagnostics GmbH. Scorpions® Probes are licensed from DxS Ltd. SYBR® and Rhodamine Green™ are trademarks and registered trademarks of Life Technologies corporation. Alexa Fluor®, BODIPY®, Cascade Blue®, Marina Blue®, Oregon Green®, Pacific Blue™,
Rhodamine Red™-X, and Texas Red®-X are trademarks and registered trademarks of and provided under an intellectual property license from Life Technologies Corporation. Black Hole Quencher® (BHQ®) is a registered trademark of Biosearch Technologies,
Inc. CAL Fluor® and Quasar® are registered trademarks of Biosearch Technologies, Inc. Locked Nucleic Acid® (LNA®) oligonucleotides produced under license from Exiqon A/S. CEQ is a trademark of Beckman Coulter, Inc. Rotor-Gene® is a registered trademark
of Qiagen N.V. MasterCycler® is a registered trademark of Eppendorf, Inc. Opticon™ and Chromo4™ are trademarks of Bio-Rad Laboratories. Mx4000®, Mx3000P®, and Mx3005P® are registered trademarks of Agilent. SmartCycler® is a registered trademark of
Cepheid, Inc. For complete label license information, please visit sigma.com/oligolicenses.
IJB
82324
1074
JB_82324_Primers and Fluorescent Probes Brochure.indd 20
9/4/14 8:26 AM