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
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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 Sigma has oligonucleotide manufacturing sites around the globe, is not just the quality of our products that sets us apart but also the in nine countries – Australia, Canada, Germany, India, Israel, Japan, quality of our service and technical expertise. 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 Manufacturing specifications are met. Our sizeable investment in state-of-the-art analytical equipment provides industry-leading tools to develop and Expertise of Our Scientists monitor our process. With more than 25 years of experience in oligonucleotide synthesis, we have the expertise to create the most technically challenging Our fluorescently-labeled probes are manufactured using a rigorous custom biomolecules. Our scientists collaborate with researchers process, including: around the world and together develop novel custom products. We • Purification by reverse phase and/or ion exchange chromatography meet customer specifications, no matter how complex. • Electrospray mass spectral analysis Quantitative PCR Tools • Quality control documentation Sigma is pleased to offer a variety of educational tools for quantitative PCR users including: • Amber packaging • Technical and troubleshooting guides Outstanding Customer Service and Technical Support PCR and qPCR technical manual Our dedicated staff of highly-trained customer service specialists are • available via email or telephone to provide timely solutions to every • Webinar series customer inquiry. Providing real-time status for orders, our customer Workshops 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.
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The MIQE Guidelines
The potential applications for quantitative real-time PCR (qPCR) By Following the MIQE Guidelines You Will: have increased exponentially since the first description (Higuchi, Promote experimental transparency 1993). However, researchers have been frustrated by complications • such as contamination, insufficient amplification, low sensitivity and • Ensure consistency between laboratories uncertainty about what constitutes a suitable statistical analysis. Until Maintain the integrity of the scientific literature recently, there has been a lack of consensus about how to handle • these obstacles. Publish or bring your product to market faster and with confidence by adhering to The MIQE Guidelines. An international research team published The MIQE (pronounced Mykee) Guidelines in 2009 to address the challenges of performing dependable qPCR measurements. To learn more about MIQE and download the paper, visit sigma.com/miqe 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
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Detection Chemistries
Sigma offers primers, probes and reagents to support all qPCR assays. There are several types of probe structures that can be used including: • Dual-Labeled Probes DNA-Binding Dyes and Probes • Molecular Beacons Quantitative real-time PCR (qPCR) relies on real-time detection of LightCycler® Probes amplification products as they are formed in the reaction. This • can be accomplished using non-specific DNA binding dyes or • Scorpions® Probes sequence-specific probes. These techniques and their benefits Each probe type enables researchers to measure an increase in are described below. fluorescent signal that corresponds to an increase in the copy number of the desired amplicon. In addition to the increase in sensitivity that A Theoretical qPCR Assay is gained from using sequence-specific probes for detection, these probes can also be labeled with different fluorescent reporter dyes, [F][F] allowing detection of multiple targets within the same PCR reaction.
thresholdthr Baseline dedetection limit 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 # There are situations in which multiplex reactions are beneficial including: 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 • Limited Template Availability: The number of amplifications can 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. be maximized Non-specific DNA Binding Dye Detection • Large Numbers of Samples: Reducing the number of reactions leads to cost savings SYBR® Green I binds non-specifically to double-stranded DNA. Upon binding, the dye undergoes a conformational change resulting However, there are a variety of challenges when developing a multi- in high fluorescent emission, allowing measurement of the total plex assay including: amount of double-stranded product present in the reaction after • Complex Design: The degree of difficulty increases with the each amplification cycle. number of targets to be detected SYBR Green I detection is a popular option due to its low cost and • Optimization Reaction Conditions: All primer/probe sets need ease of use. However, multiple double-stranded species that may be similar reaction kinetics and the same buffer, therefore target present cannot be discriminated when using SYBR Green I. Within sensitivity may be reduced compared to similar singleplex reactions any PCR, there is the potential for primer dimer formation and non- specific amplification products. Therefore, melt curves must be used Predesigned vs. Custom Assays after amplification for estimating the specificity of amplified products. Predesigned assays offer convenience since no time or effort is It may be difficult to obtain accurate quantification at low target needed for primer and probe designs or deciding on oligonucleotide concentrations. specifications, such as the right manufacturing scale, purification, For this reason, many researchers use SYBR Green I detection for yield, etc. initial screening, proof-of-concept experiments or common Sigma’s KiCqStart® Primers are ready-to-order (all specifications are applications, such as gene expression analysis, and progress to probe-based detection for greater assay sensitivity and/or for set), predesigned SYBR Green I Primers for RT-qPCR and are available multiplex analysis. for a variety of species. They can be searched and ordered online at sigma.com/ksprimers. Probe-based Detection Sometimes, demanding assays require a greater level of attention Probe-based detection methods rely on one or more fluorescently than can be found with predesigned assays. All Sigma’s detection labeled probes that are positioned between the two PCR primers. chemistries including SYBR Green I Primers, Dual-Labeled Probes, Because the probe is sequence specific, it will only detect the Molecular Beacons, LightCycler Probes, and Scorpions Probes can be presence of a single amplicon within the reaction. customized to your exact specifications.
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Assay Design
OligoArchitect™ Primer and Probe Design Solutions OligoArchitect Consultative Sigma® is pleased to offer OligoArchitect for all of your primer For more complex and demanding applications, utilize our and probe design requirements. OligoArchitect includes both consultative service to ensure the success of your assay. With personal consultation from our expert, technical support scientists, our complimentary online design tool and our unique your request, including all sequences and data analysis, will be MIQE consultative service. compliant and returned to you within 24-48 hours. If required, you will OligoArchitect Online also receive follow-up assay optimization, data analysis assistance and troubleshooting support. Requests for consultative design service can For routine needs, improve your assay with our OligoArchitect online be submitted at sigma.com/probedesignonline. design tool powered by the industry standard Beacon Designer™ (PREMIER Biosoft). The user-friendly interface utilizes the latest Designs can be completed for traditional PCR or qPCR using the algorithms, provides results in real time, supports templates up to following detection chemistries (Locked Nucleic Acid (LNA) can be 10,000 base pairs, and allows for the adjustment of input parameters included in probe and beacon designs): such as homopolymer run/repeat maximum length, G/C clamp length • SYBR Green I Primers • LightCycler Probes and maximum primer pair T mismatch. M • Dual-Labeled Probes • Scorpions Probes Designs can be completed for traditional PCR or qPCR using the • Molecular Beacons following detection chemistries (Locked Nucleic Acid® (LNA®) can be Designs can typically be completed for the following PCR and included in probe and beacon designs): qPCR applications: • SYBR® Green I Primers • LightCycler® Probes • Traditional PCR • Endpoint genotyping • Dual-Labeled Probes • Scorpions® Probes • SNP detection • Methylation analysis • Molecular Beacons • Allele discrimination • High-resolution melting analysis Our online design tool can be used for the following applications: • Pathogen detection • Transcript detection across Multiplexing exon junctions • Traditional PCR • Viral load quantification • Viral load quantification • Haplotyping • SNP detection • Gene expression analysis • Gene expression analysis • Northern blotting • Allele discrimination • Gene copy determination • Gene copy determination • Southern blotting • Pathogen detection • Endpoint genotyping • • Multiplexing Other applications: FISH (fluorescence in situ hybridization) All reported sequences, associated properties, and assay parameters • are available for export to Excel and convenient email ordering. Visit Sigma’s consultative service uses Beacon Designer from PREMIER Biosoft sigma.com/probedesignonline to try OligoArchitect Online. 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. 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.
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Reaction Optimization
Proper optimization can increase the efficiency and sensitivity of a Improved Assay Sensitivity and Specificity qPCR assay. 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 In general, amplicons should be between 50-200 bases in length. of nucleic acid analog containing a 2’-O, 4’-C methylene bridge. A Shorter amplicons tend to be more tolerant of less than ideal reaction conditions, improving the consistency of results. comparison of LNA to DNA is shown below: When quantifying RNA targets, select primers spanning exon-exon Base Base junctions to avoid amplification of contaminating genomic DNA in HO HO cDNA samples. O O
Primer and Probe Concentration O Optimization of primer and probe concentrations can improve the LNA Monomer DNA Monomer detection level of a particular amplicon by approximately 10 cycles LNA Monomer DNA Monomer depending on the sequence. Since a 3 Cq difference in amplicon When used with any standard bases (A,C,G,T, or U), probes detection indicates approximately a 10 fold difference in template synthesized using LNA have greater thermal stability than concentration, this simple step can greatly improve both the accuracy conventional DNA or RNA and therefore form a stronger bond and sensitivity of the reaction. with the complementary sequence. Primer Optimization Improves Reaction Sensitivity The introduction of LNA chemistry into a qPCR probe may result in
60 an increase in the TM of up to 8 °C per LNA monomer substitution 55 in medium salt conditions compared to a DNA fluorescent probe. It 50 45 is possible to optimize the TM level and the hybridization specificity 40 through specific placement of the LNA base(s) in the probe design as 35 Optimal
Fluorescence shown below. 30 primer concentration 25
20 Probe Sequence LNA Bases TM* ∆TM ∆TM/LNA 15 Sub-optimal 10 primer GTGATATGC 0 29 °C – – – – concentration 5 GTGATATGC 3 44 °C 15 °C +5 °C 15 20 25 30 35 40 Cycle GTGATATGC 9 64 °C 35 °C +3.9 °C
Figure 2. Primer optimization assay using Sigma SYBR® Green mastermix. Optimization of primers * of duplex between probe and its complementary sequence TM for the human UBC gene. All reactions contain exactly the same template but varying primer Note: The bolded and underlined bases denote LNA base 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 Incorporation of LNA into your qPCR probe can improve performance RT-PCR. Nature Protocols 2006; 1:1559-1582 of many assays, including: • SNP Discrimination: The presence of a single base mismatch has Buffer Conditions a greater destabilizing effect on the duplex formation between Assay performance may also benefit from optimization of buffer a LNA fluorescent probe and its target nucleic acid than with a conventional DNA fluorescent probe. components (particularly MgCl2) and the internal reference dye. Optimizing the concentration of these components is especially • Multiplex Assays: Incorporation of LNA bases allows simpler TM important when designing multiplex assays or singleplex assays in optimization, providing more flexibility in probe placement. which design of an appropriate probe/primer combination proves to be difficult. • 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.
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Data Analysis
As your qPCR experiments grow in complexity and the data volume Next Steps: becomes overwhelming, rely on our expertise to help you advance • Purchase Sigma® qPCR probes your research. • Select desirable design and analysis options • Before beginning your experiment, we will evaluate and provide recommendations for developing protocols. • Send your experimental protocols and raw data in .xls or .mdf file format to the email address below, and you will be contacted by • After completing your experiment, we will process your raw data one of our expert biostatisticians into a comprehensive results report, including all statistical analyses as well as publication-ready tables and graphs. Design and Analysis Options: • Evaluation or development of assay design 2 MDH • Identification of optimal reference genes 1.8 FBP 1.6 ADH2 • Identification of marker gene combinations HSP 1.4 SUC PGK Quantification of unknown samples PDC • 1.2 ADH1 Anti Log2) 1 TPI • Determination of statistical significance MIG
alue ( 0.8 ADH5 V ADH3 ADH6 M- 0.6 CYC Benefits: ADH4 0.4 IPPI PDA Bactin Sigma’s Biostatistics service will allow you to maintain your focus on 0.2 critical data-gathering activities while avoiding the need to purchase 0 expensive software and train personnel in time-consuming statistical Genes analyses. You will obtain results promptly so that you can publish or Figure 3. Example reference gene selection that can be included with the results report. bring your product to market faster and with confidence. To request service, send an email to [email protected]
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KiCqStart® Primers
KiCqStart Primers are predesigned SYBR® Green I Primers for two-step Guaranteed Yields and one-step RT-qPCR. Available for a variety of species, the primer Guaranteed Appx. No. Appx. No. Appx. No. pairs can be easily searched and ordered online. OD Yield of nmoles of μg of Reactions* Choose KiCqStart Primers for: 3 14 90 1,500 *Estimate based on 14 nmoles or 30 µg for 3 OD and 450 nM in a 20 uL reaction. Estimate is • Gene expression analysis based on an average sequence length of 21 bases. • Validating MISSION® siRNA and shRNA knockdowns 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) Two-step RT-qPCR with KiCqStart Primers is easy with ReadyScript® • MIQE compliant – sequences are provided at the time of shipment cDNA Synthesis Mix and KiCqStart SYBR Green qPCR ReadyMixes. Compatible with any thermal cycler • ReadyScript cDNA Synthesis Mix for Fast and Easy Preparation of Your cDNA Templates for qPCR How KiCqStart Primers Work ReadyScript cDNA Synthesis Mix is a sensitive and easy-to-use solution for two-step RT-qPCR. This 5X concentrated mix provides KiCqStart Primers are PCR primers that work with SYBR Green I. all necessary components (except RNA template) for first-strand When free in solution with only ssDNA present, SYBR Green I emits synthesis including: buffer, dNTPs, MgCl , primers, RNase inhibitor a low signal intensity. As the reaction progresses and the quantity 2 of dsDNA increases, more dye binds to the amplicons and hence, protein, ReadyScript reverse transcriptase and stabilizers. ReadyScript signal intensity increases. The illustration below depicts is a RNase H(+) derivative of MMLV reverse transcriptase, optimized for the mechanism. reliable cDNA synthesis over a wide dynamic range of input RNA. Cat. No. Product Description RDRT ReadyScript cDNA Synthesis Mix
KiCqStart SYBR Green qPCR ReadyMix™ for All Real-Time 1. Dye in solution emits 2. Emission of the PCR Instruments low fluorescence fluorescence by binding KiCqStart SYBR Green ReadyMixes for fast qPCR come in a ready-to- use 2X formulation, which includes SYBR Green I dye, an antibody- SYBR Green I cycles between an unbound (denaturation) and mediated Hot-Start Taq DNA polymerase, dNTPs, MgCl , and a bound (annealing through extension) state as the reaction 2 progresses, and signal intensity increases as the quantity of proprietary buffers and stabilizers. Just add primers and template, and amplicons increase in later cycles. you will discover that ReadyMixes offer the following features and benefits: • Speed – assay results in as little as 33 minutes Product Features Include: • Quality – highly efficient and sensitive real-time PCR results • Amount: 3 OD minimum yield • Ease of use – little to no optimization required • Purification: RP cartridge • Sequence Lengths: 18 – 24 bases Cat. No. Product Description KCQS00 KiCqStart SYBR Green qPCR ReadyMix • Quality Control: 100% mass spectrometry KCQS01 KiCqStart SYBR Green qPCR ReadyMix, Low ROX™ • Format: Supplied dry, two oligos, one per tube KCQS02 KiCqStart SYBR Green qPCR ReadyMix with ROX KCQS03 KiCqStart SYBR Green qPCR ReadyMix, iQ™
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Custom Primers
Forward and/or reverse primers are required for all custom probe Related Products and beacon detection chemistries offered by Sigma. We offer a range of products when the focus is speed and simplicity. Choose Custom Primers for: Easy Oligo: • Dual-Labeled Probes • Amount: 3 OD • Molecular Beacons • Concentration: 100 µM • LightCycler® Probes • Purification: Desalt • Scorpions® Probes • Sequence Lengths: 15 – 30 bases Benefits of Using Sigma’s Primers Include: • Quality Control: 100% mass spectrometry • Free design support with OligoArchitect • Format: Supplied in solution • Comprehensive technical support • Ships within 24 hours of order receipt • Quality guaranteed Pure & Simple Primers: Product Features Include: • Amount: 3 OD • Scale: 0.025, 0.05, 0.2, 1.0, 10, and 15 µmole • Purification: Cartridge • Purification: Desalt, Cartridge, HPLC, and PAGE • Sequence Lengths: 12 – 35 bases • Sequence Lengths: 10-120 bases (inquire for longer lengths) • Quality Control: 100% mass spectrometry • Modifications: Over 200 available, including dyes (fluorescent and • Format: Supplied dry non-fluorescent) • Ships within 24 hours of order receipt • Quality Control: 100% mass spectrometry Format: Supplied dry or in solution – tubes, plates, and mixed plates • To learn more and order, visit (forward and reverse primers) sigma.com/oligos • WellRED Oligos are also available Guaranteed Yields iScale™ Oligos Larger quantities for high-throughput projects Appx. No. • Scale (µmole) Guaranteed OD Yield Appx. No. of μg of Reactions* • Quantity: 10, 25, 50, 100, 250, 500 mg (inquire for larger quantities) 0.025 3 96 600 • Purification: Desalt, RP-HPLC, IE-HPLC 0.05 5 160 1,000 Sequence Lengths: 10 – 50 bases (inquire for longer lengths) 0.2 12 384 2,500 • 1.0 40 1,280 8,500 • Modifications: Over 200 available, including dyes (fluorescent and 10.0 400 12,800 85,000 non-fluorescent) 15.0 600 19,200 130,000 • Quality Control: 100% mass spectrometry *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. • Format: Supplied dry
To learn more and order, visit sigma.com/iscaleoligos
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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 Dual-Labeled Dual-Labeled Molecular LightCycler® Scorpions® Primers Probes LNA® Probes Beacons* Probes* 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 Several qPCR instruments utilize proprietary dyes which are not enabling flexibility in the choice of probe labels. It is important to generally available commercially, such as VIC™ and NED™. When select the fluorescent labels which are compatible with the detection seeking dye alternatives, the following criteria are important: channels for the qPCR instrument and to ensure the correct filter • The excitation and detection wavelength are compatible with the settings or detection calibration for the instrument. The Reporters instrument light source and detection system and Instrument Compatibility table (see page 11) lists a selection of For probes, the quencher effectively absorbs light at the emission some of the most widely used qPCR platforms and indicates which • wavelength of the reporter fluorescent labels may be used. Please note that not all labels are listed and many alternative reporters are available. For information on • The higher the extinction coefficient the brighter the dye, which the use of non-standard labels with these platforms, contact your local contributes to sensitive detection technical service professional ([email protected]). • 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.
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Spectral Properties Table Dye Max. Max. Compatible Dye Max. Max. Compatible EX (nm) EM (nm) Quencher EX (nm) EM (nm) Quencher 6-FAM™ 494 515 BHQ-1, TAMRA Cyanine 3 550 570 BHQ-2 JOE™ 520 548 BHQ-1, TAMRA Quasar® 5703 548 566 BHQ-2 TET™ 521 536 BHQ-1, TAMRA Cal Fluor Red 5904 565 588 BHQ-2 Cal Fluor® Gold 5401 522 541 BHQ-1 ROX™ 573 602 BHQ-2 HEX™2 535 555 BHQ-1, TAMRA Texas Red® 583 603 BHQ-2 Cal Fluor Orange 5602 540 561 BHQ-1 Cyanine 5 651 674 BHQ-3 TAMRA™ 555 576 BHQ-2 Quasar 6705 647 667 BHQ-3 1JOE/TET alternative 3Cyanine 3 alternative 5Cyanine 5 alternative Cyanine 5.5 675 694 BHQ-3 2VIC® alternative 4TAMRA alternative 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® FAM HEX JOE ROX TET Cyanine Cyanine TAMRA Texas Red LC Red 640 LC Red Green I 3 5 705 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.
JB_82324_Primers and Fluorescent Probes Brochure.indd 11 9/4/14 8:26 AM JB_82324_Primers andFluorescent Probes Brochure.indd 12 to thatemitanywhere utilize reporters inthisrange. itpossible 3 cover from thespectrum 480nminto thenearIRmaking andDual-LabeledProbes.Beacons As shown inFigure 2,and 5,BHQ-1, efficiencies, itanexcellent whichmakes quencherfor Molecular provides quenching asignificant increase inbothstaticandFRET withtheemissionmaximaofFAM,superimposable and JOE. TET This athigherwavelengthsnm) isabsorbed thanDABCYL andisdirectly maximumof534 Black HoleQuencher1(BHQ®-1)(withanabsorption quenching). quencher thatutilize FRET Dual-Labeled Probes (linearprobes and withseparated reporter shown inFigurethe reporters 4,therefore itisapoorchoicefor maximumof474nmplacesitbelow themaximaof its absorption with paired andquencherthatutilize staticquenching), reporter Though DABYCL (structured probes Beacons well works inMolecular RFU =relative fluorescence unit. 6-FAM™,reporters andJOE™.DABCYL wasnormalized atthepoly-TTET™, (260nm). absorbance Figure ofDABCYL-T9 spectra 4.Absorption ofthecommonly-used withtheemissionspectra dyes emittingabove withreporter poorly 480nm(Figurevery 4). quencher,a dark footprint hasaninadequate absorption thatoverlaps background becauseofitsown native fluorescence. DABCYL, though quencher andtherefore to contributes anoverall increase in ofqPCR. andflexibility ultimate sensitivity TAMRA isnotadark Two commonlyusedquenchers, TAMRA™ andDABYCL, limitthe Black HoleQuencher bionucleics Abs (DABCYL-T9) Abs RFU (JOE, TET, FAM) 250 250 300 300 T9 DABCYL-T9 350 350 BHQ-1 BHQ-2 BHQ-3 T9 400 400 6-F TE JOE Wa (534 nm) (579 nm) (672 nm) 450 T Wa 450 AM v elength (nm) v elength (nm) 500 500 6-F
S:N 550 TET 550 AM (518) 7.5 9.0 1.5 3.0 4.5 6.0 0.0 HEX (553)/JOE(554) (538) Cyanine 3(565) 600 600 T AMRA (583) ROX (607) TAM 650 LC Red(640) 650 5’-FAM Probes (667) Cyanine 5
DAB 3’ Quencher 700 700 ® 750 750 BHQ-1 BHQ-2 or TAMRA probes (Figure 6). signal-to-noise ratioswhencompared to thecorresponding DABCYL additionto overlap, better spectral In BHQprobes have muchhigher TET, HEX™,Cyanine 3, TAMRA, ROX™, LC 640,andCyanine Red 5. poly-T 6-FAM, of260nm)withtheemissionmaximacommonly-usedreporters absorbance Figure ofthethree (conjugated spectra BHQvariants to 5.Absorption T-9 andnormalized to the fluorescence oftheprobe intensity alone. target sequence by the 25mer inthepresence excesscomplementary ofafive-fold ofanexact Figure 6.Signal-to-noise(S:N)ratioswere calculated by dividingthefluorescence signal ofa Abs (DABCYL-T9) RFU (JOE, TET, FAM) S:N Abs 80 250 20 40 60 0 250 300 5’-Cyanine 3Probes 300 DABB T9 DABCYL-T9 3’ Quencher S:N 350 7.5 9.0 350 1.5 3.0 4.5 6.0 0.0 BHQ-1 BHQ-2 BHQ-3 HQ-1 T9 400 400 TAM 6-F TE JOE Wa BHQ-2 (534 nm) (579 nm) (672 nm) 450 T Wa 450 AM v 5’-FAM Probes elength (nm) v DAB 3’ Quencher elength (nm) 500 500
S:N 6-F 550 BHQ-1 TET 120 550 AM (518) 30 60 90 0 HEX (553)/JOE(554) (538) Cyanine 3(565) 600 600 T 5’-Cyanine 5Probes AMRA (583) BHQ-2 DABB ROX (607) 3’ Quencher 650 LC Red(640) 650 (667) Cyanine 5
HQ-2 700 700
S:N 80 750 20 40 60 BHQ-3 0 750
5’-Cyanine 3Probes
DABB 9/4/14 8:26 AM 3’ Quencher HQ-1 BHQ-2
S:N 120 30 60 90 0 5’-Cyanine 5Probes DABB 3’ Quencher HQ-2 BHQ-3 sigma.com/probes 13
Finally, as shown in Figure 7, the BHQ variants readily permit single- In Summary, Black Hole Quencher: tube multiplexing due to the increased variety of reporters that can 1) has no native fluorescence (emits heat instead of light), resulting be effectively quenched with little or no cross-talk. This simplifies the in lower background fluorescence design, implementation, and interpretation of multiplexed assays. 2) has increased signal-to-noise ratio, providing higher sensitivity 3) enables a wider choice of reporters for multiplexing
6-FAM TET JOE Normalized Emission
450 500 550 600 650 700 750 Wavelength (nm)
6-FAM TAM ROX CYANINE 5 Normalized Emission
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.
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Dual-Labeled Probes
Dual-Labeled Probes are the most common probe type for qPCR and Product Features Include: are often referred to as hydrolysis probes. • Amounts: 1, 3, 5, and 10 OD Choose Dual-Labeled Probes for: • Purification: HPLC • Sequence Lengths: 15 - 40 bases • Microarray validation • Multiplexing • Quality Control: 100% mass spectrometry • Multiple target genes / • Viral load quantification • Format: Supplied dry in amber tubes few samples • Gene expression analysis • Custom formats available (normalization, special plates, etc.) Pathogen detection Gene copy determinations • • Sigma’s probes are provided in a format to simplify your experimental planning. Perfect for validating MISSION® siRNA and shRNA knockdowns Guaranteed Yields Benefits of Using Dual-Labeled Probes Include: Guaranteed Appx. No. Appx. No. Appx. No. • Design simplicity for sequence specificity OD Yield of nmoles of μg of Reactions* • Extensive availability of reporter/quencher combinations 1 4 32 800 • Increased sensitivity 3 12 96 2,400 Add LNA® to Your Probe for: 5 20 160 4,000 • Increased thermal stability and hybridization specificity 10 40 320 8,000 • Greater accuracy in SNP detection, allele discrimination, and in vitro * Estimate is based on 4 nmoles or 32 μg for 1 OD and 200 nM in a 25 µL reaction quantification or detection (5.0 pmol/reaction). Estimate is based on an average sequence length of 25 bases. • Easier and more sensitive probe designs for problematic The most common reporter and quencher combinations are target sequences listed below: Spectral Properties Table How Dual-Labeled Probes Work Dye Max. EX Max. EM Compatible (nm) (nm) Quencher A Dual-Labeled Probe is a single-stranded oligonucleotide labeled with two different dyes. A reporter dye is located at 6-FAM™ 494 515 BHQ®-1, TAMRA™ the 5’ end and a quencher molecule located at the 3’ end. The JOE™ 520 548 BHQ-1, TAMRA quencher molecule inhibits the natural fluorescence emission of TET™ 521 536 BHQ-1, TAMRA the reporter by fluorescence resonance energy transfer (FRET). 1 The illustration below depicts the mechanism. Cal Fluor® Gold 540 522 541 BHQ-1 HEX™2 535 555 BHQ-1, TAMRA 2 5’ Reporter Cal Fluor Orange 560 540 561 BHQ-1 R TAMRA™ 555 576 BHQ-2
5’ Reporter Cyanine 3 550 570 BHQ-2 3’ Quencher 3’ Quencher R Taq Quasar® 5703 548 566 BHQ-2 Q DNA Q polymerase Cal Fluor Red 5904 565 588 BHQ-2
Amplified Target DNA ROX™ 573 602 BHQ-2 Texas Red® 583 603 BHQ-2 1. Probe in solution emits 2. Emission of the fluorescence low fluorescence by hydrolysis Cyanine 5 651 674 BHQ-3 The primer is elongated by the polymerase and the probe binds Quasar 6705 647 667 BHQ-3 to the specific DNA template. Hydrolysis releases the reporter Cyanine 5.5 675 694 BHQ-3 from the probe/target hybrid, causing an increase in fluorescence. 1JOE/TET alternative 3Cyanine 3 alternative 5Cyanine 5 alternative The measured fluorescence signal is directly proportional to the 2VIC® alternative 4TAMRA alternative amount of target DNA. 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.
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Molecular Beacons
Molecular Beacons are structured probes that are highly sensitive, Product Features Include: sequence specific, and are used for sequence detection in qPCR and • Amounts: 1, 3, 5, and 10 OD in vitro studies. • Purification: HPLC Choose Molecular Beacons for: • Sequence Lengths: 15 - 40 bases • Quality Control: 100% mass spectrometry • SNP detection • Gene expression analysis • Format: Supplied dry in amber tubes • Allele discrimination • Gene copy determination • Custom formats available (normalization, special plates, etc.) • Pathogen detection • End point genotyping • Multiplexing • In vitro quantification or Sigma’s probes are provided in a format to simplify your experimental • Viral load quantification detection planning.
Benefits for Using Molecular Beacons Include: Guaranteed Yields Probe preserved during the reaction • Guaranteed Appx. No. Appx. No. Appx. No. • Increased specificity OD Yield of nmoles of μg of Reactions* Add LNA to Your Probe for: 1 3 32 600 • Increased thermal stability and hybridization specificity 3 9 96 1,800 • Greater accuracy in SNP detection, allele discrimination, and in vitro 5 15 160 3,000 quantification or detection 10 30 320 6,000 • Easier and more sensitive probe designs for problematic target * Estimate is based on 3 nmoles or 32 μg for 1 OD and 200 nM in a 25 µL reaction (5.0 pmol/ sequences reaction). Estimate is based on an average sequence length of 30 bases.
The most common reporter and quencher combinations are How Molecular Beacons Work listed below: A Molecular Beacon is a single-stranded bi-labeled fluorescent probe held in a hairpin-loop conformation (around 20 to 25 nt) by Dye Max. EX Max. EM Compatible complementary stem sequences (around 4 to 6 nt) at both ends (nm) (nm) Quencher of the probe. The 5’ and 3’ ends of the probe contain a reporter 6-FAM™ 494 515 BHQ-1, DABCYL and a quencher molecule, respectively. The loop is a single- Fluorescein 495 520 BHQ-1, DABCYL stranded DNA sequence complementary to the target sequence. JOE™ 520 548 BHQ-1, DABCYL The close proximity of the reporter and quencher causes the quenching of the natural fluorescence emission of the reporter. TET 521 536 BHQ-1, DABCYL The structure and mechanism of a Molecular Beacon is shown HEX 535 555 BHQ-1, DABCYL below. Cyanine 3 550 570 BHQ-2, DABCYL,
Loop ROX™ 573 602 BHQ-2, DABCYL Sequence Loop Sequence Texas Red® 583 603 BHQ-2, DABCYL, 5’ Reporter Cyanine 5 651 674 BHQ-3, DABCYL, Stem 3’ Quencher Sequence R Q Cyanine 5.5 675 694 BHQ-3, DABCYL,
Q R 5’ Reporter Amplified Target DNA Sigma® is a licensed supplier of a variety of dyes and quenchers and 3’ Quencher continually adds to its portfolio of new chemistries. For assistance in 1. Unbound beacon with 2. Bound beacon with the design of your Molecular Beacon assays, use our online design quenched fluorescence unquenched fluorescence tool at sigma.com/probedesignonline. 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.
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LightCycler® Probes
LightCycler probes are highly sensitive and sequence-specific Add LNA® to Your Probe for: fluorescent probes designed for use with the Roche LightCycler • Increased thermal stability and hybridization specificity instruments. • Greater accuracy in SNP detection and allele discrimination Choose LightCycler Probes for: • Easier and more sensitive probe designs for problematic target sequences • SNP detection • Viral load quantification Product Features Include: • Allele discrimination • Gene expression analysis • Pathogen detection • Gene copy determination • Amounts: 0.1, 0.25, 1.5, and 15 OD • Multiplexing • Purification: HPLC • Sequence Lengths: 15 - 40 bases Several reporters are available and are suitable for multiplex analysis. • Quality Control: 100% mass spectrometry • Format: Supplied dry in amber tubes Benefit of Using LightCycler Probes include: • Custom formats available (normalization, special plates, etc.) • Increased specificity Sigma’s probes are provided in a format to simplify your Probe preserved during the reaction • experimental planning. • Increased thermal stability and hybridization specificity • Greater accuracy in SNP detection and allele discrimination • Easier and more sensitive probe designs for problematic Guaranteed Yields of LightCycler Probes target sequences Guaranteed Appx. No. Appx. No. Appx. No. OD Yield of nmoles of μg of Reactions* How LightCycler Probes Work 0.1 0.4 3.2 80 0.25 1 8 200 A LightCycler Probe system consists of a pair of single-stranded fluorescent-labeled oligonucleotides. Oligo Probe 1 is labeled at 1.5 6 48 1,200 the 3’ end with a donor reporter and Oligo Probe 2 is labeled at 15 60 480 12,000 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 * 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. group (P) to prevent DNA polymerase extension. There should be a space of 1 to 5 nt to separate the two probes from each The recommended constructs for Oligo Probe 1 and Oligo Probe 2 are other. The structures and mechanism of a LightCycler probe are listed in the tables below: shown below. Labels and Modifications for LightCycler Probes 3’ Donor Fluorophore (FD)
FD FD P FA Oligo Probe 1 FRET Oligo Probe 1 5’ Acceptor 3’ end Fluorescein Fluorophore (FA) Oligo Probe 2* FA P Amplified Target DNA Oligo Probe 2 3’ end Phosphate 5’ end LightCycler Red 610, 640, 670 and 705 1. Probes in solution emit 2. Emission through fluorescence low fluorescence resonance energy transfer *For enhanced discrimination, LNA can be incorporated into Probe 2 During the annealing step of qPCR, the PCR primers and the LightCycler Probes hybridize to their specific target regions Sigma® is a licensed supplier of a variety of dyes and quenchers and bringing the donor dye into close proximity to the acceptor dye. continually adds to its portfolio of new chemistries. For assistance in When the donor dye is excited by light from the LightCycler the design of your LightCycler Probe assays, use our online design tool instrument, energy is transferred by FRET from the donor to at sigma.com/probedesignonline. 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.
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Scorpions® Probes
Scorpions probes are highly sensitive, sequence specific, bi-labeled Add LNA to Your Probe for: fluorescent probe/primer hybrids designed for qPCR. • Increased thermal stability and hybridization specificity Choose Scorpions for: • Greater accuracy in SNP detection and allele discrimination • Easier and more sensitive probe designs for problematic • SNP detection • Viral load quantification target sequences Allele discrimination Gene expression analysis • • Product Features Include: • Pathogen detection • Gene copy determination • Multiplexing • Endpoint genotyping • Amounts: 1, 5, and 10 OD • Purification: HPLC Because the probe and primer are incorporated into a single • Sequence Lengths: 30 - 60 bases (Uni-Probe) and 15 - 45 bases molecule, the reaction kinetics of this probe are extremely fast. The (Bi-Probe) reaction leading to generation of a fluorescent signal is essentially • Quality Control: 100% mass spectrometry instantaneous and occurs prior to any competing side reactions. This Format: Supplied dry in amber tubes enables Scorpions Probes to provide stronger signals, shorter reaction • Custom formats available (normalization, special plates, etc.) times and better discrimination than other conventional bi-molecular • mechanisms. It also allows for more reliable probe design. Sigma’s probes are provided in a format to simplify your experimental planning. Benefits of Using Scorpions Probes Include: Guaranteed Yields • Increased specificity • Fast amplicon detection Guaranteed Appx. No. Appx. No. Appx. No. • Exceptional signal-to-noise (bi-probes typically yield a stronger OD Yield of nmoles of μg 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 * 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). fluorescent probe sequence held in a hairpin-loop conformation with a 5’ end reporter and an internal quencher directly linked to The available reporter and quencher combinations are listed below. the 5’ end of a PCR primer via a blocker. The blocker prevents the Scorpions Probes include a HEG (hexathylene glycol) blocker. polymerase from extending the PCR primer. Uni-Probe Loop Loop Blocker Sequence 5’ Reporter Internal Quencher Sequence Internal Quencher 5’ Reporter Fluorescein, 6-FAM™, HEX™, TET™, TAMRA™, DABCYL dT, Stem Sequence JOE™, ROX™, Cyanine 3, Cyanine 5, Cyanine 5.5, BHQ®-1, Q Internal Texas Red®, Rhodamine, Rhodamine Green™, BHQ-2, Quencher R R Q Rhodamine Red™, Oregon Green® 488, Oregon BHQ-3 PCR Primer PCR Primer 5’ Reporter Newly Synthesized Green 500, Oregon Green 514 DNA Strand Blocker Target DNA Complementary Sequence Bi-Probe 1. Quenching of the fluorescence 2. Emission of the fluorescence 5’ Reporter 3’ Quencher At the beginning of the qPCR, the polymerase extends the PCR Fluorescein, 6-FAM, HEX, TET, TAMRA, JOE, ROX, TAMRA, DABCYL, primer and synthesizes the complementary strand of the specific Cyanine 3, Cyanine 5, Cyanine 5.5, Texas Red, BHQ-1, target sequence. During the next cycle, the hairpin-loop unfolds Rhodamine, Rhodamine Green, Rhodamine BHQ-2, and the loop-region of the probe hybridizes intramolecularly to Red, Oregon Green 488, Oregon Green 500, BHQ-3 the newly synthesized target sequence. Now that the reporter Oregon Green 514 is no longer in close proximity to the quencher, fluorescence emission may take place. The fluorescent signal is detected by the Sigma® is a licensed supplier of a variety of dyes and quenchers and qPCR instrument and is directly proportional to the amount of continually adds to its portfolio of new chemistries. For assistance in target DNA. the design of your Scorpions Probe assays, use our online design tool at sigma.com/probedesignonline. To learn about the bi-probe mechanism, please visit sigma.com/ probes, and click Learn More under Scorpions Probes.
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WellRED Primers
Sigma is licensed by Beckman Coulter, Inc., to supply WellRED Yields and Estimated Number of Assays dye-labeled oligos designed for use with the CEQ™/GeXP Genetic Analysis Systems and may be used for direct hybridization or in PCR Yield (OD) 1 2 4 5 10 amplification. WellRED dye-labeled oligos are DNA oligonucleotides Purification PAGE Cartridge Cartridge HPLC HPLC labeled with WellRED dyes, D2, D3 or D4, at the 5’ end. Approx. yield 5 10 20 25 50 (nmol*) Estimated assays 500-1,000 1,000- 2,000- 2,500- 5,000- WellRED Specifications (reactions) 2,000 4,000 5,000 10,000
Yields Cartridge: 2 and 4 OD *Estimate 1 OD = 5 nmol = 30 µg, for a 20 base oligo HPLC: 5, 10, 50 and 100 OD PAGE: 1 OD Purification Cartridge, HPLC, PAGE Spectral Properties of WellRED Dyes Length 7 – 100 bases Excitation Emission Molar extinction Backbone Phosphodiester WellRED Oligo maximum (nm) maximum (nm) coefficient Format Supplied Dry D2-PA 750 770 170,000 Modifications D2-PA, D3-PA or D4-PA at the 5’ end D3-PA 685 706 224,000 Packaging 2 mL Opaque Tubes D4-PA 650 670 203,000 Quality Controls MALDI-TOF MS, Electrospray Ionization MS, OD by UV Spectroscopy To learn more and order, visit Technical Datasheet & Includes yield, Tm, MW, and µg/OD Labels Duplicate set of labels with each order sigma.com/wellred Additional Services Aliquoting into tubes MSQC 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.
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OligoEvaluator™
Sigma is pleased to offer OligoEvaluator, our online oligonucleotide OligoEvaluator™ sequence calculator.
Functionality Module Features Analysis 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 OligoEvaluator Input Inferface to check for problematic folding Check for oligonucleotide sequence homology with BLAST • All reported properties are available for export to a convenient Excel Algorithm template. Your OligoArchitect™ login can be used for OligoEvaluator. To try OligoEvaluator, visit sigma.com/oligocalculator. • Powered by PREMIER Biosoft
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©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
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