Primers and Fluorescent Probes - Sigma-Aldrich: Analytical

KiCqStart® Primers

Custom Primers

Black Hole Quencher®

Dual-Labeled Probes

Molecular Beacons

LightCycler® Probes

Scorpions® Probes

WellRED Primers

bionucleics

Primers and Fluorescent ProbesFor Quantitative Real-Time PCR and other Applications

JB_82324_Primers and Fluorescent Probes Brochure.indd 1 9/4/14 8:26 AM

bionucleics

Predesigned and Custom ProductsSigma 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 AdvantageWe 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.

The Sigma advantage includes:

• Our Commitment to Quality

• Outstanding Customer Service and Technical Support

• Global Manufacturing

Our Commitment to QualityQuality 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 satisfac-tion. 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.

Global ManufacturingSigma 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.

Sigma® Life Science Custom ProductsA Custom Solution for Every Project – Guaranteed

Manufacturing

Expertise of Our ScientistsWith 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 ToolsSigma 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


sigma.com/probes 3

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: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

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

The MIQE Guidelines


Detection ChemistriesSigma offers primers, probes and reagents to support all qPCR assays.

DNA-Binding Dyes and ProbesQuantitative 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.

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 ReactionsThe use of probes labeled with different reporter dyes allows the simultaneous detection and quantification of multiple target genes in a single (multiplex) reaction.

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 multi-plex 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 AssaysPredesigned 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.

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.

A Theoretical qPCR Assay

Non-specific DNA Binding Dye DetectionSYBR® 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 non-specific 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 DetectionProbe-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.

threshold

[F]

Baseline

Cq Cq Cq Cq

Cycle #

detection limit

thr

de

[F]

Baseline

bionucleics


sigma.com/probes 5

Assay DesignOligoArchitect™ Primer and Probe Design SolutionsSigma® 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.

OligoArchitect OnlineFor 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

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.

OligoArchitect ConsultativeFor 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.

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 ServicesWith 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. 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.

• Traditional PCR• SNP detection• Allele discrimination• Pathogen detection• Multiplexing

• Viral load quantification• Gene expression analysis• Gene copy determination• Endpoint genotyping

Our online design tool can be used for the following applications:


bionucleics

Reaction OptimizationProper optimization can increase the efficiency and sensitivity of a qPCR assay.

Primer and Probe PlacementIn 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 and Probe ConcentrationOptimization 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.

Improved Assay Sensitivity and Specificity Using Locked Nucleic Acid® (LNA®)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:

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

Primer Optimization Improves Reaction Sensitivity

Buffer ConditionsAssay 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.

60

55

50

45

40

35

Flu

ore

scen

ce

30

25

20

15

15 20 25Cycle

30 35 40

10

5

Optimalprimer

concentration

Sub-optimal primer concentration

LNA Monomer

HOBase

O

O

DNA Monomer

HOBase

O

Probe Sequence 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 sequenceNote: The bolded and underlined bases denote LNA base

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.

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.

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 TM optimization, providing more flexibility in probe placement.

• 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.

LNA Monomer DNA Monomer


IPPI PDA Bactin

ADH4CYC

ADH6ADH3

ADH5MIG

TPIADH1

PDCPGK

SUCHSP

ADH2FBP

MDH

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

Genes

M-V

alue

(Ant

i Log

2)

sigma.com/probes 7

Data AnalysisAs 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.

• After completing your experiment, we will process your raw data into a comprehensive results report, including all statistical analyses as well as publication-ready tables and graphs.

Next Steps:• 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

Design and Analysis Options:• 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: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]

Figure 3. Example reference gene selection that can be included with the results report.


bionucleics

KiCqStart® PrimersKiCqStart 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.

Choose KiCqStart Primers for:

• Gene expression analysis

• Validating MISSION® siRNA and shRNA knockdowns

Benefits of Using KiCqStart Primers Include:

• Developed with sophisticated bioinformatics tools and validated in 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

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

Guaranteed OD Yield

Appx. No. of nmoles

Appx. No. of μg

Appx. No. of Reactions*

3 14 90 1,500

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.

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.

Guaranteed Yields

*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 sigma.com/ksprimers

Companion Products

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. Product DescriptionRDRT 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-to-use 2X formulation, which includes SYBR Green I dye, an antibody-mediated 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. Product DescriptionKCQS00 KiCqStart SYBR Green qPCR ReadyMix

KCQS01 KiCqStart SYBR Green qPCR ReadyMix, Low ROX™

KCQS02 KiCqStart SYBR Green qPCR ReadyMix with ROX

KCQS03 KiCqStart SYBR Green qPCR ReadyMix, iQ™

1. Dye in solution emits low fluorescence

2. Emission of the fluorescence by binding


sigma.com/probes 9

Custom PrimersForward and/or reverse primers are required for all custom probe and beacon detection chemistries offered by Sigma.

Choose Custom Primers for:

• Dual-Labeled Probes

• Molecular Beacons

• LightCycler® Probes

• Scorpions® Probes

Benefits of Using Sigma’s Primers Include:

• Free design support with OligoArchitect

• Comprehensive technical support

• Quality guaranteed


• Scale: 0.025, 0.05, 0.2, 1.0, 10, and 15 µmole

• Purification: Desalt, Cartridge, HPLC, and PAGE

• Sequence Lengths: 10-120 bases (inquire for longer lengths)

• Modifications: Over 200 available, including dyes (fluorescent and non-fluorescent)


• Format: Supplied dry or in solution – tubes, plates, and mixed plates (forward and reverse primers)

• WellRED Oligos are also available

Guaranteed Yields

Scale (µmole) Guaranteed OD Yield Appx. No. of μg

Appx. No.

of Reactions*0.025 3 96 600

0.05 5 160 1,000

0.2 12 384 2,500

1.0 40 1,280 8,500

10.0 400 12,800 85,000

15.0 600 19,200 130,000* 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.

Related ProductsWe offer a range of products when the focus is speed and simplicity.

Easy Oligo:

• Amount: 3 OD

• Concentration: 100 µM

• Purification: Desalt



• Format: Supplied in solution

• Ships within 24 hours of order receipt

Pure & Simple Primers:

• Amount: 3 OD

• Purification: Cartridge



• Format: Supplied dry

• Ships within 24 hours of order receipt

To learn more and order, visit sigma.com/oligos

iScale™ Oligos

• 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)


• Format: Supplied dry

To learn more and order, visit sigma.com/iscaleoligos


bionucleics

Choosing the Right Custom Probe

Reporters, Quenchers and Instrument CompatibilityModern 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]).

Dye SubstitutesSeveral 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

QuenchersQuenching 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.

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.


sigma.com/probes 11

Spectral Properties Table

Dye Max. EX (nm)

Max. EM (nm)

Compatible Quencher

6-FAM™ 494 515 BHQ-1, TAMRA

JOE™ 520 548 BHQ-1, TAMRA

TET™ 521 536 BHQ-1, TAMRA

Cal Fluor® Gold 5401 522 541 BHQ-1

HEX™2 535 555 BHQ-1, TAMRA

Cal Fluor Orange 5602 540 561 BHQ-1

TAMRA™ 555 576 BHQ-21JOE/TET alternative 3Cyanine 3 alternative 5Cyanine 5 alternative 2VIC® alternative 4TAMRA alternative

Dye Max. EX (nm)

Max. EM (nm)

Compatible Quencher

Cyanine 3 550 570 BHQ-2

Quasar® 5703 548 566 BHQ-2

Cal Fluor Red 5904 565 588 BHQ-2

ROX™ 573 602 BHQ-2

Texas Red® 583 603 BHQ-2


Quasar 6705 647 667 BHQ-3

Cyanine 5.5 675 694 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 probe and/or assays, visit sigma.com/probedesignonline.

Reporters and Instrument Compatibility Table

Platform SYBR® Green I

FAM HEX JOE ROX TET Cyanine 3

Cyanine 5

TAMRA Texas Red LC Red 640 LC Red 705

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 • • • • • •Qiagen Rotor-Gene® 6000 • • • • • • • • •Eppendorf Mastercycler® ep realplex • • • • • • •

Note: Not all qPCR instruments or reporters are listed. Contact the instrument manufacturer for details on compatible reporters.


Abs

(DA

BCYL

-T9)

RFU

(JO

E, T

ET, F

AM

)

250 300 350 400 450 500 550 600 650 700 750

Wavelength (nm)

JOETET6-FAM

T9

DABCYL-T9

Abs

Wavelength (nm)

250 300 350 400 450 500 550 600 650 700 750

T9

6-FAM (518)TET (538)

HEX (553)/JOE (554)Cyanine 3 (565)

TAMRA (583)ROX (607)

LC Red (640)Cyanine 5 (667)

BHQ-3 (672 nm)BHQ-2 (579 nm)BHQ-1 (534 nm)

bionucleics

Black Hole Quencher®

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).

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.

Though DABYCL works well in Molecular Beacons (structured probes with paired reporter and quencher that utilize static quenching), its absorption maximum of 474 nm places it below the maxima of the reporters shown in Figure 4, therefore it is a poor choice for Dual-Labeled Probes (linear probes with separated reporter and quencher that utilize FRET quenching).

Black Hole Quencher 1 (BHQ®-1) (with an absorption maximum of 534 nm) is absorbed at higher wavelengths than DABCYL and is directly superimposable with the emission maxima of FAM, TET and JOE. This provides a significant increase in both static and FRET quenching efficiencies, which makes it an excellent quencher for Molecular Beacons and Dual-Labeled Probes. As shown in Figure 5, BHQ-1, 2, and 3 cover the spectrum from 480 nm into the near IR making it possible to utilize reporters that emit anywhere in this range.

Abs

(DA

BCYL

-T9)

RFU

(JO

E, T

ET, F

AM

)

250 300 350 400 450 500 550 600 650 700 750

Wavelength (nm)

JOETET6-FAM

T9

DABCYL-T9

Abs

Wavelength (nm)

250 300 350 400 450 500 550 600 650 700 750

T9

6-FAM (518)TET (538)

HEX (553)/JOE (554)Cyanine 3 (565)

TAMRA (583)ROX (607)

LC Red (640)Cyanine 5 (667)

BHQ-3 (672 nm)BHQ-2 (579 nm)BHQ-1 (534 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.

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).

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.

0.0

1.5

3.0

4.5

6.0

7.5

9.0

TAM DAB BHQ-1

S:N

3’ Quencher

5’-FAM Probes

BHQ-20

20

40

60

80

DAB BHQ-1

S:N

3’ Quencher

5’-Cyanine 3 Probes

BHQ-20

30

60

90

120

DAB BHQ-2

S:N

3’ Quencher


BHQ-3

0.0

1.5

3.0

4.5

6.0

7.5

9.0

TAM DAB BHQ-1

S:N

3’ Quencher

5’-FAM Probes

BHQ-20

20

40

60

80

DAB BHQ-1

S:N

3’ Quencher


BHQ-20

30

60

90

120

DAB BHQ-2

S:N

3’ Quencher


BHQ-3


sigma.com/probes 13

Finally, as shown in Figure 7, the BHQ variants readily permit single-tube 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.

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.

Nor

mal

ized

Em

issi

on

450 500 550 600 650 700 750

Wavelength (nm)

6-FAMTETJOE

Nor

mal

ized

Em

issi

on

450 500 550 600 650 700 750

Wavelength (nm)

6-FAMTAMROXCYANINE 5

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 sensitivity3) enables a wider choice of reporters for multiplexing


bionucleics

Dual-Labeled ProbesDual-Labeled Probes are the most common probe type for qPCR and are often referred to as hydrolysis probes.

Choose Dual-Labeled Probes for:


• 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.

Dye Max. EX (nm)

Max. EM (nm)

Compatible Quencher

6-FAM™ 494 515 BHQ®-1, TAMRA™

JOE™ 520 548 BHQ-1, TAMRA

TET™ 521 536 BHQ-1, TAMRA

Cal Fluor® Gold 5401 522 541 BHQ-1

HEX™2 535 555 BHQ-1, TAMRA

Cal Fluor Orange 5602 540 561 BHQ-1

TAMRA™ 555 576 BHQ-2


Quasar® 5703 548 566 BHQ-2

Cal Fluor Red 5904 565 588 BHQ-2

ROX™ 573 602 BHQ-2

Texas Red® 583 603 BHQ-2


Quasar 6705 647 667 BHQ-3

Cyanine 5.5 675 694 BHQ-3

Guaranteed OD Yield

Appx. No. of nmoles

Appx. No. of μg


1 4 32 800

3 12 96 2,400

5 20 160 4,000

10 40 320 8,000

1JOE/TET alternative 3Cyanine 3 alternative 5Cyanine 5 alternative 2VIC® alternative 4TAMRA alternative

How Dual-Labeled Probes Work

Amplified Target DNA

5’ Reporter

5’ Reporter

TaqDNApolymerase

3’ Quencher 3’ Quencher

1. Probe in solution emits low fluorescence

2. Emission of the fluorescence by hydrolysis

R Q Q

R

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.

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.

Guaranteed Yields

* 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 most common reporter and quencher combinations are listed below:

Spectral Properties Table

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.

Perfect for validating MISSION® siRNA and shRNA knockdowns

Benefits of Using Dual-Labeled Probes Include:

• Design simplicity for sequence specificity• Extensive availability of reporter/quencher combinations• Increased sensitivity

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• Easier and more sensitive probe designs for problematic

target sequences

• Microarray validation • Multiple target genes /

few samples• Pathogen detection

• Multiplexing• Viral load quantification• Gene expression analysis• Gene copy determinations


sigma.com/probes 15

Molecular BeaconsMolecular 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:

Benefits for Using Molecular Beacons Include:

• Probe preserved during the reaction• Increased specificity

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• Easier and more sensitive probe designs for problematic target

sequences

How Molecular Beacons Work

Dye Max. EX (nm)

Max. EM (nm)

Compatible 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.


5’ Reporter

5’ Reporter

3’ Quencher

3’ Quencher

1. Unbound beacon with quenched fluorescence

2. Bound beacon with unquenched fluorescence

Loop Sequence

Stem Sequence

Loop Sequence

R

RQ

Q

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 single-stranded 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.

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.


• 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

Guaranteed OD Yield

Appx. No. of nmoles

Appx. No. of μg


1 3 32 600

3 9 96 1,800

5 15 160 3,000

10 30 320 6,000* 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.

• SNP detection• Allele discrimination• Pathogen detection• Multiplexing• Viral load quantification

• Gene expression analysis• Gene copy determination • End point genotyping• In vitro quantification or

detection

The most common reporter and quencher combinations are listed below:


bionucleics

LightCycler® ProbesLightCycler probes are highly sensitive and sequence-specific fluorescent probes designed for use with the Roche LightCycler instruments.

Choose LightCycler Probes for:

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

target sequences

The recommended constructs for Oligo Probe 1 and Oligo Probe 2 are listed in the tables below:

Labels and Modifications for LightCycler Probes

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.

How LightCycler Probes Work


5’ Acceptor Fluorophore (FA)

FRETOligo Probe 1

Oligo Probe 2

3’ Donor Fluorophore (FD)

1. Probes in solution emit low fluorescence

2. Emission through fluorescence resonance energy transfer

FDFD FA

FA P

P

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.

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.

Guaranteed Yields of LightCycler Probes

Guaranteed OD Yield

Appx. No. of nmoles

Appx. No. of μg


0.1 0.4 3.2 80

0.25 1 8 200

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.

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

• SNP detection • Allele discrimination• Pathogen detection• Multiplexing

• Viral load quantification• Gene expression analysis• Gene copy determination

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


• 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.


sigma.com/probes 17

Scorpions® ProbesScorpions probes are highly sensitive, sequence specific, bi-labeled fluorescent probe/primer hybrids designed for qPCR.

Choose Scorpions for:


• 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.

The available reporter and quencher combinations are listed below. Scorpions Probes include a HEG (hexathylene glycol) blocker.

How Uni-Probe Scorpions Probes Work

Target DNA

PCR Primer

5’ Reporter

5’ Reporter

Stem Sequence

Internal Quencher

Internal Quencher

Blocker

Blocker

LoopSequence

Complementary Sequence

Newly SynthesizedDNA Strand

PCR Primer

Loop Sequence

1. Quenching of the fluorescence 2. Emission of the fluorescence

RR

Q

Q

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.

Guaranteed OD Yield

Appx. No. of nmoles

Appx. No. of μg


1 2 32 400

5 10 160 2,000

10 20 320 4,000

Guaranteed Yields

Uni-Probe

Bi-Probe

* 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).

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

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.

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

signal when compared to uni-probes)

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• SNP detection• Allele discrimination• Pathogen detection • Multiplexing

• Viral load quantification• Gene expression analysis• Gene copy determination • Endpoint genotyping

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.


bionucleics

WellRED PrimersSigma 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 Cartridge: 2 and 4 ODHPLC: 5, 10, 50 and 100 ODPAGE: 1 OD

Purification Cartridge, HPLC, PAGELength 7 – 100 basesBackbone PhosphodiesterFormat Supplied DryModifications D2-PA, D3-PA or D4-PA at the 5’ endPackaging 2 mL Opaque TubesQuality Controls MALDI-TOF MS, Electrospray Ionization MS,

OD by UV SpectroscopyTechnical Datasheet & Labels

Includes yield, Tm, MW, and µg/ODDuplicate set of labels with each order

Additional Services Aliquoting into tubesMSQC traces available upon request

Shipping Schedule Shipped within 4 to 5 business days

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.

Yields and Estimated Number of Assays

Yield (OD) 1 2 4 5 10Purification PAGE Cartridge Cartridge HPLC HPLCApprox. yield (nmol*)

5 10 20 25 50

Estimated assays (reactions)

500-1,000 1,000-2,000

2,000-4,000

2,500-5,000

5,000-10,000

Spectral Properties of WellRED Dyes

WellRED OligoExcitation maximum (nm)

Emission maximum (nm)

Molar extinction coefficient

D2-PA 750 770 170,000D3-PA 685 706 224,000D4-PA 650 670 203,000

*Estimate 1 OD = 5 nmol = 30 µg, for a 20 base oligo

To learn more and order, visit sigma.com/wellred


OligoEvaluator Input Inferface

OligoEvaluator™

Sigma is pleased to offer OligoEvaluator, our online oligonucleotide sequence calculator.

Functionality

Module FeaturesAnalysis 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)

Resuspension Enter the quantity, desired concentration, and molecular weight, and the tool returns the volume of water or buffer needed to resuspend a dry oligonucleotide

Dilution 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 to check for problematic folding

• Check for oligonucleotide sequence homology with BLAST

Algorithm

• Powered by PREMIER Biosoft

sigma.com/probes 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.

OligoEvaluator™


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

Sigma-Aldrich® Worldwide Offices

IJB823241074

©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.

World Headquarters 3050 Spruce St.

St. Louis, MO 63103 (314) 771-5765

sigma-aldrich.com

ArgentinaFree Tel: 0810 888 7446 Tel: (+54) 11 4556 1472 Fax: (+54) 11 4552 1698

AustraliaFree Tel: 1800 800 097 Free Fax: 1800 800 096 Tel: (+61) 2 9841 0555 Fax: (+61) 2 9841 0500

AustriaTel: (+43) 1 605 81 10 Fax: (+43) 1 605 81 20

BelgiumFree Tel: 0800 14747 Free Fax: 0800 14745 Tel: (+32) 3 899 13 01 Fax: (+32) 3 899 13 11

BrazilFree Tel: 0800 701 7425 Tel: (+55) 11 3732 3100 Fax: (+55) 11 5522 9895

CanadaFree Tel: 1800 565 1400 Free Fax: 1800 265 3858 Tel: (+1) 905 829 9500 Fax: (+1) 905 829 9292

ChileTel: (+56) 2 495 7395 Fax: (+56) 2 495 7396

ChinaFree Tel: 800 819 3336 Tel: (+86) 21 6141 5566 Fax: (+86) 21 6141 5567

Czech RepublicTel: (+420) 246 003 200 Fax: (+420) 246 003 291

DenmarkTel: (+45) 43 56 59 00 Fax: (+45) 43 56 59 05

FinlandTel: (+358) 9 350 9250 Fax: (+358) 9 350 92555

FranceFree Tel: 0800 211 408 Free Fax: 0800 031 052 Tel: (+33) 474 82 28 88 Fax: (+33) 474 95 68 08

GermanyFree Tel: 0800 51 55 000 Free Fax: 0800 64 90 000 Tel: (+49) 89 6513 0 Fax: (+49) 89 6513 1160

HungaryIngyenes telefonszám: 06 80 355 355 Ingyenes fax szám: 06 80 344 344 Tel: (+36) 1 235 9063 Fax: (+36) 1 269 6470

IndiaTelephone 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

IrelandFree Tel: 1800 200 888 Free Fax: 1800 600 222 Tel: (+353) 402 20370 Fax: (+ 353) 402 20375

IsraelFree Tel: 1 800 70 2222 Tel: (+972) 8 948 4100 Fax: (+972) 8 948 4200

ItalyFree Tel: 800 827 018 Tel: (+39) 02 3341 7310 Fax: (+39) 02 3801 0737

JapanTel: (+81) 3 5796 7300 Fax: (+81) 3 5796 7315

Korea Free Tel: (+82) 80 023 7111 Free Fax: (+82) 80 023 8111 Tel: (+82) 31 329 9000 Fax: (+82) 31 329 9090

MalaysiaTel: (+60) 3 5635 3321 Fax: (+60) 3 5635 4116

MexicoFree Tel: 01 800 007 5300 Free Fax: 01 800 712 9920 Tel: (+52) 722 276 1600 Fax: (+52) 722 276 1601

The NetherlandsFree Tel: 0800 022 9088 Free Fax: 0800 022 9089 Tel: (+31) 78 620 5411 Fax: (+31) 78 620 5421

New ZealandFree Tel: 0800 936 666 Free Fax: 0800 937 777 Tel: (+61) 2 9841 0555 Fax: (+61) 2 9841 0500

NorwayTel: (+47) 23 17 60 00 Fax: (+47) 23 17 60 10

PolandTel: (+48) 61 829 01 00 Fax: (+48) 61 829 01 20

PortugalFree Tel: 800 202 180 Free Fax: 800 202 178 Tel: (+351) 21 924 2555 Fax: (+351) 21 924 2610

RussiaTel: (+7) 495 621 5828 Fax: (+7) 495 621 6037

SingaporeTel: (+65) 6779 1200 Fax: (+65) 6779 1822

SlovakiaTel: (+421) 255 571 562 Fax: (+421) 255 571 564

South AfricaFree Tel: 0800 1100 75 Free Fax: 0800 1100 79 Tel: (+27) 11 979 1188 Fax: (+27) 11 979 1119

SpainFree Tel: 900 101 376 Free Fax: 900 102 028 Tel: (+34) 91 661 99 77 Fax: (+34) 91 661 96 42

SwedenTel: (+46) 8 742 4200 Fax: (+46) 8 742 4243

SwitzerlandFree Tel: 0800 80 00 80 Free Fax: 0800 80 00 81 Tel: (+41) 81 755 2828 Fax: (+41) 81 755 2815

United KingdomFree Tel: 0800 717 181 Free Fax: 0800 378 785 Tel: (+44) 1747 833 000 Fax: (+44) 1747 833 313

United StatesToll-Free: 800 325 3010 Toll-Free Fax: 800 325 5052 Tel: (+1) 314 771 5765 Fax: (+1) 314 771 5757

VietnamTel: (+84) 3516 2810 Fax: (+84) 6258 4238

Internet sigma-aldrich.com

Enabling Science to Improve the Quality of Life


Documents

Primers and Fluorescent Probes - Sigma-Aldrich: Analytical