A revolution in PCR analysis!

Fast, convenient qualitative and quantitative PCR analysis Detect amplicon in seconds Generate accurate quantitative gene expression data Eliminate post-PCR processing Minimize risk of contamination with closed-well detection Ideal for high-throughput multiplexing Sentinel™ Molecular Beacon System for PCR Analysis Applications How the System Works: Sentinel molecular beacons provide a new method for PCR Determination of target presence/absence analysis that is faster and more convenient than ever before: Quantitative gene expression analysis 1. Construct a molecular beacon probe specific for Positive endogenous controls your target. 2. Add the molecular beacon probe to the PCR mixture. Design custom assays 3. Run PCR. As PCR proceeds, product accumulates and the molecular beacon produces fluorescence pro- Allele discrimination portional to the amount of specific amplicon present. Single nucleotide mutation-detection 4. Monitor the reaction by measuring fluorescence either PCR multiplexing as the reaction is occurring or when cycling is complete. Qualitative vs. Quantitative Fluorescence OUT WITH THE OLD: Gel electrophoresis, Measurements with Molecular Beacons semi-quantitative data Sentinel molecular beacons are well suited for both qualita- PCR is typically followed by slow, tedious analysis using gel tive (endpoint) and quantitative (real-time) PCR analysis and electrophoresis. Mutagenic ethidium bromide has to be monitoring. Either may be used for the most basic molecular handled cautiously. Tubes have to be opened to remove beacon application, which is determining the presence of a samples, increasing the risk of carry-over contamination. specific amplified product. The amount of quantitative data gathered by gel analysis is Qualitative PCR analysis utilizes a fluorometer such limited. Now there is a better way to analyze your PCR. as Stratagene’s FluorTracker™ fluorescence reader, which is IN WITH THE NEW: PCR analysis with molecular designed to accommodate all PCR labware (tubes, strips and beacons, truly quantitative data 96-well PCR plates) and optimized for molecular beacon ™ fluorescence. Qualitative PCR analysis produces accurate PCR analysis with Sentinel molecular beacons opens the results rapidly allowing a single fluorescence reader to easily door to true quantitative analysis while saving time and work handle the output of a number of thermal cyclers. When by completely eliminating electrophoresis and the need for your PCR is completed, you are only minutes from discover- post-PCR manipulation. Analyzing your PCR has never ing if your amplicon is present. Quantitative assessments been so quick or easy. based on endpoint fluorescence, however, are inherently Better than gel electrophoresis inaccurate due to the influences of limiting reagents and and other fluorescent PCR systems small differences in reaction components or cycling Faster and easier than electrophoresis parameters. Truly quantitative PCR analysis Quantitative PCR analysis requires a spectrofluoro- Minimized risk of carry-over contamination metric thermal cycler. Using molecular beacons to monitor PCR reactions in real time provides quantitative data for Most sensitive PCR system for single nucleotide mismatch detection and allele-discrimination gene expression analysis and accurate determination of Versatile cycling parameters simplify initial copy number. primer/probe design Each cycle produces a fluorescent signal proportional to High-throughput and multiplexing capabilities the amount of amplicon present. Results are typically Higher specificity than systems using fluorescent displayed on an amplification plot showing the change in DNA-binding dyes or primers fluorescence with cycle number. This information may be used to quantify initial target copy number. Studies have shown that initial copy number can be accurately quantified during PCR by determining threshold cycle, that cycle at which the amplification plot crosses a defined fluorescence threshold. Since the threshold cycle is inversely proportional to the log of the initial copy number, the more template that is initially present the lower the cycle number where the fluorescence exceeds the threshold.

Molecular Target Hybrid Beacon Sentinel™ Molecular Beacon Core Reagent Kits Single-Tube RT-PCR Core Reagent Kit For quantitative and qualitative PCR analysis Single-tube convenience and speed Fast and convenient Analysis of gene expression accurate over a wide range Exceptional sensitivity of target inputs Great versatility The single-tube RT-PCR core reagent kit includes all the reagents Supports multiplexing and high-throughput formats necessary to quickly and conveniently carry out cDNA synthesis and The Sentinel™ molecular beacon core reagent kits** provide all the PCR PCR amplification in one tube for RNA analysis using molecular and RT-PCR components needed for creating custom molecular beacon beacon detection. The convenient and fast single-tube format reduces assays. Moreover, these kits are specifically designed for use with the the risk of carry-over contamination. Sentinel molecular beacon detection and expression analysis kits. This kit is sufficient for 200 reactions and includes a kit control. The kit is designed for use with the Sentinel molecular beacon PCR Core Reagent Kit expression analysis kits and custom molecular beacon-based assays.

Detects a wide variety of DNA targets 8000 Mouse Total RNA (pg) 7000 Wide linear range of amplification 105 6000 104 The Sentinel molecular beacon PCR core reagent kit includes all the 5000 103 components necessary to carry out PCR amplification. This kit may 4000 102 3000 10 be used in combination with Stratagene’s molecular beacon probe kits 1 2000 No Target or custom designed molecular beacons to amplify and detect a wide 1000 Control

Fluorescence Intensity No RT variety of DNA targets, including genomic DNA, cDNA and plasmid 0 Threshold -1000 DNA. 0102030405152535 This kit is sufficient for 200 reactions and includes a kit control. A Cycle Number Standard Curve The kit is designed for use with the Sentinel molecular beacon 40 detection kits, expression analysis kits and custom molecular beacon- 35 based assays. 30 Ct 25 19000 Copies of Plasmid DNA 17000 108 20 15000 107 15 13000 106 11000 10 2 3 4 5 5 1 10 10 10 10 10 9000 10 B Total RNA (pg) 7000 104 5000 103 3000 2 Wide Linear Dynamic Range and Great Sensitivity

Fluorescence Intensity 10 1000 Amplification and detection of mouse ␤-actin message from mouse total RNA using single-tube RT-PCR No Target 5 -1000 Control core reagents. Amplification plot shows a target titration from 1-10 pg mouse total RNA resulting in 0 10203040Threshold threshold cycle (Ct) values from 35 to 19 respectively (Figure A). When plotted as Ct versus the log of target Cycle Number A input, the resulting standard curve is linear over 6 orders of magnitude with a regression correlation of 0.9963 (Figure B). Standard Curve 50 40 RT-PCR Core Reagent Kit 30 Ct 20 Superior sensitivity 10 Analysis of gene expression accurate over a wide range 0 of target inputs 10 102 103 104 105 106 107 108 109 The RT-PCR core reagent kit is formatted for a traditional two-tube B Copies of Plasmid DNA RT-PCR reaction and includes all the reagents necessary to carry out Wide Linear Dynamic Range of Amplification cDNA synthesis and PCR amplification for RNA analysis using Amplification and detection of plasmid DNA using PCR core reagents. Amplification plot shows a target titration from 102 to 108 copies of plasmid DNA resulting in threshold cycle (Ct) values from 35 to 15 molecular beacon detection. This format allows versatility and the respectively (Figure A). When plotted as Ct versus the log of target input, the resulting standard curve is possibility of running additional controls. The RT and PCR reactions linear over 7 orders of magnitude with a regression correlation of 0.9962 (Figure B). are optimized to provide exceptional sensitivity. This kit is sufficient for 200 reactions and includes a kit control. The kit is designed for use with the Sentinel molecular beacon Custom Molecular Beacon Synthesis expression analysis kits and custom molecular beacon-based assays. Stratagene offers a custom synthesis service for molecular beacon Abundance of GAPDH mRNA Relative to ␤-Actin mRNA fluorescent . 1.40 High-quality molecular beacons Single Tube 1.20 Multi-step purification process Two Tube Quality assurance data sent with every probe 1.00 Multiple fluorophores available (6-FAM, TET, HEX, Cy3, Cy5, TAMRA, Texas Red® and others by special order) 0.80 Signal-to-noise and thermal denaturation profile available 0.60 These dual-labeled fluorescent oligonucleotides may be synthe- 0.40 sized with a variety of dyes. They are shipped in a highly purified Relative Abundance form with quality assurance data, including HPLC trace, 0.20 0.00 molecular weight and quantity (O.D. units and nanomoles). When Liver Brain Stomach an order for a custom molecular beacon is combined with an order Tissue Type for a complementary nucleotide target, the signal-to-noise ratio Great Reproducibility Using the Single-Tube or Two-Tube RT-PCR Kits and thermal denaturation profile for the beacon-target pair is also The abundance of GAPDH mRNA relative to ␤-actin mRNA is equivalent using either the supplied. single-tube or two-tube RT-PCR formats. To order a custom molecular beacon fluorescent or for more information, please contact Stratagene’s Technical Support at 800-894-1304. Sentinel™ Molecular Beacon FluorTracker™ Fluorescence Reader Detection Kits For endpoint PCR analysis For use as endogenous controls Economical fluorometer for detecting presence of PCR products Optimized for PCR plates, tubes and strips Compatible with human, rat and mouse templates Great sensitivity ␤ Available for -Actin or GAPDH Easily accommodates Use either genomic DNA or cDNA templates simultaneous output of multiple Evaluate new molecular beacon systems thermal cyclers Flexible temperature parameters Separate thermal cycling and Qualitative or quantitative PCR analysis detection for high-throughput and economy ™ Sentinel molecular beacon detection kits* provide positive endog- Ideal for allele discrimination enous control reactions for use in determining the presence of a Detects a variety of fluorophores specific nucleotide sequence. These kits may also be used to deter- mine initial amount of target when using genomic DNA templates or The FluorTracker™ fluorescence reader provides fast, automated to evaluate newly designed molecular beacon systems using this well- detection (qualitative endpoint assay) of PCR amplification products characterized control system. in conjunction with Sentinel™ molecular beacon kits. With this easy- These detection kits recognize PCR product generated from to-use fluorometer, samples are simply transferred directly to the human, mouse and rat genomic DNA and cDNA templates. FluorTracker reader following thermal cycling for automatic measure- Each kit contains the specific molecular beacon and primer mix for ment. Since the PCR samples are transferred without being opened, 100 reactions, and includes a human genomic control template. These the risk of contamination by amplicons is minimized. kits are designed for use with the Sentinel molecular beacon core Software. The Windows®-based software interface provides precision reagent kits. control and maximum flexibility with point-and-click selection of filters, gain values, timing parameters and other functions. Microsoft® 1.600 Human Genomic DNA Excel performs data reduction, graphic analysis and unlimited 1.400 1000 ng 1.200 100 ng customized calculations. 1.000 10 ng 0.800 FAM Filters 1 ng ® Rn FAM and Texas Red ⌬ 0.600 100 pg Texas Red Filters 0.400 10 pg 13.00 0.200 1 pg 0.000 No Template 11.00 -0.200 024 6810 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 9.00 Cycle Number 7.00 Great Sensitivity and Wide Linearity The GAPDH detection kit demonstrates both great sensitivity and linearity using a serial dilution of human Sig/NTC 5.00 genomic DNA. The standard curve for this data set (not shown) is linear over 6 orders of magnitude with a regression correlation of 0.9975. 3.00 1.00 MBs + FAM target MBs + TR target MBs + both targets Well Contents ™ Sentinel Molecular Beacon Detection of FAM and Texas Red® Fluorophores The FluorTracker fluorescence reader easily detects and discriminates multiple fluorophores. Each tube Expression Analysis Kits contains the FAM- and Texas Red-labeled molecular beacons and the target sequence as indicated. The y- For quantitative gene expression analysis axis represents the fluorescence signal of the sample divided by the signal obtained from the no-target control. Highly specific for cDNA Sensitive RT-PCR analysis Sensitivity. Liquid light guide technology and a high-energy xenon ™ flash lamp provide broad spectrum excitation. Combined with the Sentinel molecular beacon expression analysis kits* are highly specific system’s corresponding range of emission and excitation filters, this for cDNA due to the design of the molecular beacon and primer pair. technology provides the sensitivity and versatility needed to collect This ensures highly accurate quantitative results. Stratagene offers a data from experiments using a wide variety of fluorophores. An variety of expression analysis kits that include probes for control/ effective noise reduction system eliminates the influence of ambient housekeeping targets and targets useful for investigating gene light and PMT dark current to give the FluorTracker reader unsur- expression. passed sensitivity. Each kit contains the specific molecular beacon, primer mix for 100 reactions and a control template. These kits are specifically Any microplate format. The customized plate holder accommo- designed for use with the Sentinel molecular beacon core reagent kits. dates PCR microplates, tubes and strips. By defining the locations of the corner wells, any microplate format may be read with minimal crosstalk. 8000 123456 7000 6000 differentiated Sentinel™ Molecular Beacon 5000 4000 Allele Discrimination Kits 3000 Superior sensitivity 2000 undifferentiated 1000 In a single PCR reaction, genotyping a specific polymorphism is Fluorescence Intensity 0 performed by using two differentially labeled molecular beacons for wild-type and mutant alleles. The hairpin shape of the molecular beacon causes mismatched probe/target hybrids to easily dissociate at 0246810 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 significantly lower temperatures than a perfectly complementary Cycle Number molecular beacon. The resulting increase in sensitivity makes Quantifying aP2 Gene Expression molecular beacons the ideal choice for allele discrimination. The quantification of expression is easily determined between differentiated THP-1 cells, with and without Each kit contains two allele-specific molecular beacons, se- stimulation (lanes 1-3 undifferentiated; lanes 4-6 PMA differentiated; lanes 1, 4 unstimulated; lanes 2, 5 quence-matched DNA templates and PCR primers. LPS stimulated; lanes 3, 6 INF-␥ stimulated), and is supported by observation of the RT-PCR products on an agarose gel. Sentinel™ Molecular Beacons Technology

MOLECULAR BEACON How Molecular Beacons Work

MOLECULAR BEACON HYBRID

DNA

TARGET

A molecular beacon is a with a fluorophore When the complementary target is present, the molecular beacon and a non-fluorescent quencher molecule at opposite ends of an spontaneously binds to this target. This binding causes a change oligonucleotide. The ends of the oligonucleotide are designed to in the conformation of the probe that places the fluorophore and be complementary to each other and form a stem structure while quencher far enough apart that the fluorophore is no longer the intervening loop is designed to be complementary to a quenched and the molecular beacon fluoresces. sequence within the amplified product. When the molecular The fluorescence of the molecular beacon is monitored during beacon is free in solution, it adopts a hairpin structure which PCR or after cycling is complete. The amount of fluorescence brings the fluorophore and quencher sufficiently close to each indicates the amount of specific product present at that time. other to allow quenching of the fluorophore. Molecular beacons Real-time analysis monitors PCR at each cycle, allowing accurate whose loop sequence is complementary to the product amplified quantitation. Endpoint analysis detects the presence of a particular from the gene of interest are added to PCR reactions along with sequence when PCR cycling is complete. all the other PCR components.

PCR with Molecular Beacons

At denaturation temperatures, all nucleotide pairs are dissociated, and molecular beacons fluoresce At extension temperatures, brightly. the molecular beacons dissociate from the target, ensuring that the molecular beacon does not interfere with polymerization.

At annealing temperatures, appropriately designed molecular beacons hybridize to specific targets that are exact complements and the amount of fluorescence is proportional to the amount of product. If the target DNA sequence does not exactly match the molecular beacons sequence,

Temperature even if only by a single base pair, hybridization and fluorescence will not occur. The thermodynamic properties of When PCR is completed, at room temperature molecular beacons are such that they are or 4˚C the molecular beacons remain bound to more stable in the hairpin form than the specific target and fluorescence is hybridized to a less than perfectly matched proportional to the amount of specific DNA sequence. amplicon present.

DENATURATION ANNEALING EXTENSION ENDPOINT AND ANALYSIS REAL-TIME ANALYSIS Ordering Information Catalog No. Sentinel™ Custom Molecular Beacon Synthesis Call for Information or visit www.stratagene.com Molecular Sentinel™ Molecular Beacon Core Reagent Kits Beacon Kits PCR Core Reagent Kit 200 rxns 600500 Single-Tube RT-PCR Core Reagent Kit 200 rxns 600505 and RT-PCR Core Reagent Kit 200 rxns Coming Soon FluorTracker™ Sentinel™ Molecular Beacon Detection Kits ␤-Actin Detection Kit 100 rxns 200570 Fluorescence GAPDH Detection Kit 100 rxns 200571 Sentinel™ Molecular Beacon Expression Analysis Kits Reader Human ␤-Actin Expression Analysis Kit 100 rxns Coming Soon Mouse ␤-Actin Expression Analysis Kit 100 rxns 200573 Human GAPDH Expression Analysis Kit 100 rxns 200575 Human TNF-␣ Expression Analysis Kit 100 rxns 200525 Human ␤-2 Microglobulin Expression Analysis Kit 100 rxns 200526 Human aP2 Expression Analysis Kit 100 rxns 200528 Sentinel™ Molecular Beacon Allele Discrimination Kits CCR2 Allele Discrimination Kit Coming Soon Factor V Allele Discrimination Kit Coming Soon FluorTracker™ Fluorescence Reader and Accessories FluorTracker™ Fluorescence Reader 120V 401230 Includes reader, PCR plate and tube holder, two filter sets (FAM, Texas Red/ROX) 100V 401231 230V 401232 Allele-Calling Software for use with FluorTracker™ Fluorescence Reader Coming Soon Install on your computer. Requires Windows® 95. Custom Filter Sets for the FluorTracker™ Fluorescence Reader Call for Information Please contact Stratagene Technical Services

These Molecular Beacons products are sold under license from The Public Health Research Institute of The City of New York, Inc. for the STRATAGENE USA and CANADA purchaser’s own internal research and development activities. ORDER: (800) 424-5444 x3 *Purchase of these PCR-related products does not convey any rights under the PCR patents owned by Hoffmann-La Roche. A license to TECHNICAL SERVICES: 800-894-1304 use the PCR process accompanies the purchase of certain reagents from Stratagene when used in conjunction with an Authorized Thermal Cycler. STRATAGENE EUROPE **Purchase of this product is accompanied by a license to use in the Polymerase Chain Reaction (PCR) process in conjunction with an Belgium, France, Germany, Authorized Thermal Cycler. Stratagene’s PCR products are sold under licensing arrangements with Roche Molecular Systems, Inc., F. The Netherlands, Switzerland, Hoffman-La Roche and the Perkin-Elmer Corporation. United Kingdom Texas Red® is a registered trademark of Molecular Probes, Inc. ORDER: 00800 7000 7000 TECHNICAL SERVICES: 00800 7400 7400 Austria ORDER: 0800 312 526 TECHNICAL SERVICES: 017 956 7036

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