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C A T A L O G U E Proteomics • Genomics Biologics • Systems • Quantitative and qualitative PCR Technology All types of probes and related expertise in one company

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Page 1: Real Quant PCR

C A T A L O G U E

Proteomics •

Genomics •

Biologics •

Systems •

Quantitative and qualitative PCR Technology

All types of probes and related expertise

in one company

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TABLE

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NTS Quantitative and qualitative

PCR TechnologyINTRODUCTION 4

INSTRUMENTATION 5

• Flexible instruments 5

- Smart Cycler® 5

- Capillary system 6

- Corbett Rotor-Gene 6

• High-throughput systems 7

- ABI Prism™ Sequence Detection Systems 7

- ICycler™ 7

- MX-4000™ Multiplex Quantitative 7

- DNA Engine Opticon™ 7

TECHNOLOGIES 8

• Introduction 8

• Non specific detection systems 9

• Specific detection systems 9

- Double-Dye Oligonucleotide probes 10

- Eclipse™ probes 12

- Molecular Beacon probes 13

- Scorpions™ probes 15

- Hybridization probes 16

• Other technologies 17

- ResonSense® probes 17

- Light-up probes 17

- Hy-Beacon™ probes 18

• Quenching and FRET 18

- Proximal quenching 18

- FRET quenching 18

- Examples 19

• New Quencher 20

- The ElleQuencher™ 20

- The Eclipse™ Dark Quencher 21

2

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• Chemical structure of the most common quenchers 22

• Dyes and quenchers chemistry 23

APPLICATIONS 26

• Quantitative PCR 26

- Relative quantification 27

- Absolute quantification 28

• Qualitative PCR 28

PRIMER AND PROBE DESIGN 29

• Introduction 29

• Double-Dye Oligonucleotide design 29

• Scorpions™ design 30

• Molecular Beacon design 31

• Hybridization probe design 32

PROTOCOLS 33

FREQUENTLY ASKED QUESTIONS 35

EUROGENTEC PRODUCTS 37

• Kits and consumables 37

- For 96-well plate systems 37

- For capillary system 37

- For Smart Cycler® 38

- Consumables 38

• R2T2 reverse transcriptase kits 38

- For instruments requiring passive reference 38

- For Smart Cycler® 39

• Fluorescent probes 39

- Molecular Beacon probes 39

- Double-Dye Oligonucleotide probes 40

- Scorpions™ probes 40

- FRET probes for capillary system 40

• Fluorescent dyes and quenchers 40

• Smart Cycler® equipment 41

JARGON 42

REFERENCES 44

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Copyright © 2002 Eurogentec s.a., Parc scientifique du Sart Tilman, 4102 Seraing, Belgium. All rightsreserved under International Copyright Convention. No part of this book may be reproduced in anyform whatsoever without written permission from the publisher.

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ON Introduction

Qualitative and quantitative (Q&Q) PCR is a method that has been introducedrecently. This technology combines DNA amplification with detection of theproducts in a single tube. The homogeneous format is highly beneficial as itremoves the significant contamination risk caused by opening tubes for post-PCRmanipulation. It is also less time consuming than gel based analysis and cansupply a qualitative and quantitative result.

Current detection methods are based on changes in fluorescence proportional tothe increase in product, whether specific or non-specific. Fluorescence is monitoredduring each PCR cycle to provide an amplification plot, allowing the user tofollow the reaction (Figure 1).

There are now a number of qualitative and quantitative PCR (Real-Time PCR)machines designed to carry out Real-Time reactions and analyse the results.With the introduction of easy to use reagent kits, Q&Q PCR has become anattractive option for applications such as quantitative PCR and allelicdiscrimination.

This document aims to provide a comprehensive, simple guide to Q&Q PCR,covering the different technologies used, their applications, design, protocols foruse and troubleshooting. For a quick description of many of the terms that you willencounter and to find a useful troubleshooting guide, see the end of this document.

There are a number of decisions to be made once the initial decision to use Q&Q PCR has been made for a particular qualitative or quantitative application.Firstly, there is the choice of instrument and chemistry. Many of the machinesare capable of running most of the current chemistries but care should be takenwhen matching the machine with chemisry. The alternative to purchasing a Q&QPCR machine is to contract out the work to one of a growing number ofcompanies running Real-Time services.

Cycles

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Figure 1: Q&Q PCR amplification plot. Units on the axes are x=cycles, y=fluorescence. Plotis taken from the ABI Prism™ 7700 Sequence Detection Software.

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NInstrumentationThere are currently several qualitative and quantitative PCR machines on themarket. They could be classified in two different categories in function of theneeds of the user: flexible instruments and high-throughput instruments.

The flexible instruments are systems more dedicated for smaller batches ofsamples but are faster and present more flexibility, allowing the user to rundifferent parameters each time.

The high-throughput instruments will be dedicated for laboratories running largebatches of samples and few different parameters. They have a 96 or 384 well-plate format.

FLEXIBLE INSTRUMENTS

Smart Cycler®

The Smart Cycler® System from Cepheid is a highly versatile and efficient thermalcycler with Real-Time optical detection, specifically tailored to the rapidlyevolving needs of today’s molecular biology laboratory.

The Smart Cycler® is based on an original concept, the modular one. The coreof the Smart Cycler® System is the I-CORE® (Intelligent Cooling/Heating OpticalReaction) module, incorporating state-of-the-art microfluidic and microelectronicdesign. Each Smart Cycler® processing block contains sixteen independentlyprogrammable I-CORE modules, each of which performs four-color, Real-Timefluorometric detection. Samples are amplified and measured in the SmartCycler®’s proprietary sealable reaction tubes, which are designed to optimizerapid thermal transfer and optical sensitivity. Six blocks can be linked togetherto give a high-throughput machine.

The Smart Cycler® software enables single or multiple operators to define andsimultaneously carry out any number of separate experiments, each with aunique set of cycling protocols, threshold criteria, and data analysis.

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N In addition, thermal and optical data from each and all sites can be monitoredin real time, and graphs of temperature, growth curves, and melt curves can becharted as the data are collected. This will increase considerably the throughputof the system, and will be ideal for optimization of different cycling conditions.

The Smart Cycler® is an open system being able to run all existing chemistries asSYBR Green™ I, Double-Dye Oligonucleotide, Molecular Beacons, Scorpions™probes and FRET technology.

It is capable of exciting and detecting 4 different dyes at once (Multiplexing)using several LED’s and silicon photodetectors.

The following websites give information on the Smart Cycler®:www.eurogentec.com and www.oswel.com

Capillary system

This instrument is very similar to the Idaho Rapid Cycler, from which it wasdeveloped. Rather than using plastic tubes and plates this machine uses glasscapillaries. Consequently, specialised master mixes for the capillary systems arerequired as some reagents stick to the glass. This is discussed further in thesection on protocols. The capillary system is extremely fast, with a ramp rate of20 °C per second, but is only capable of doing 32 samples in one run. Dyeexcitation is from a single LED at 470 nm and it only detects at 530 nm (FAM or SYBR Green™ I), 640 nm and 710 nm. There are very few dyes that are excitedat 470 nm and emit at 640 or particularly 710 nm. This causes a problem formultiplexing probes. The machine was originally set up for hybridisation probes,which use FRET (Fluorescence resonance energy transfer) (Ju et al. 1995) totransfer energy from FAM to the Roche dyes LC red 640 and LC red 705, asdiscussed further below. This machine is also suitable for fast cycling using aFAM Double-Dye Oligonucleotide probe or SYBR Green™ I. There is a novelScorpions™ and Molecular Beacon design that also allows detection of ROX inchannel 2 (640 nm).

Corbett Rotor-Gene

The Corbett Rotor-Gene combines a Q&Q PCR machine with a centrifuge. Thesamples are held in a rotor, which spins at high speed and eliminates all sampleto sample variation in temperature. The rotor takes 32 or 72 samples. There are4 excitation sources; LED’s emitting at 470 nm, 530 nm, 585 nm and 625 nm.It has 6 detection channels ranging from 510 nm-660 nm and there should beno spectral overlap between the channels. This machine can detect a wide rangeof dyes.

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NHIGH-THROUGHPUT SYSTEMS

ABI Prism™ 7000, 7700 and 7900Sequence Detection Systems

These are high-throughput machines (96-well plate format for the 7000/7700and 96 or 384-well plate format for the 7900), with the 7900 being sold witha robotic plate loading system. They are often called Double-DyeOligonucleotide machines as they were developed for the Double-DyeOligonucleotide technology discussed below. Excitation of the fluorescent dyesis via a laser. Dyes that emit between 500-660 nm can be detected. Thestandard setup is such that the common dyes used are FAM, TET, JOE, VIC™ (formore information, see pages 11-12 and 36), ROX, TAMRA and SYBR Green™I (all dye names will be given as the standard abbreviation rather than fullchemical name). Protocols take 2-3 hours.

ICycler™

The iCycler™ from Bio-Rad is a 96-well block PCR machine that is a standardPCR machine with an optical unit attached. The unit uses a CCD detector todetect emission from each well simultaneously and can excite and detect 4different dyes at once.

MX-4000™ Multiplex Quantitative

The MX4000™ Multiplex Quantitative PCR System from Stratagene is a high-throughput machine using 96-well plates. It uses a halogen lamp for excitationbetween 350-750 nm. It can detect 4 fluorophores (350 nm-830 nm) in onereaction and different excitation and emission filter sets are available. These canbe customised for each user.

DNA Engine Opticon™

The DNA engine Opticon™ from MJ Research is a Real-Time system based onthe PTC 200 thermal cycler. It combines a 96-well DNA Engine™ and an opticalsystem, the Opticon™ fluorescence detector. An array of 96 blue LEDs illuminatethe cycler wells one at a time, and a photomultiplier tube detects fluorescence.

The DNA Engine Opticon™ is a single-colour device compatible with populardye chemistries including SYBR Green™ I dye, Double-Dye Oligonucleotideprobes and Molecular Beacons.

The Opticon excites fluorescent dyes with absorption spectra in the 450 to 495nm range. The system is optimised for dyes with emission spectra in the 515 to545 nm range.

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S Quantitative and qualitativePCR TechnologiesINTRODUCTION

To understand how Q&Q technologies work it is first necessary to understandPCR (Figure 2). In brief, this technology was first developed in 1983 to amplifyfragments of DNA. It uses the ability of a thermo-stable DNA polymerase enzyme(taq) to extend short single stranded synthetic oligonucleotides (primers) duringrepeated cycles of heat denaturation, primer annealing and primer extension.The primers are designed to bind to the DNA fragment to be amplified. The taquses the target DNA added to the reaction as a template for primer extension.Each cycle more DNA is synthesised, providing additional template. Thereaction proceeds in an exponential manner, doubling the amount of target eachcycle, until one of the reagents becomes limiting and the reaction reaches aplateau.

There are 2 types of DNA detection chemistries that are used in qualitative andquantitative PCR. Specific sequence detection distinguishes between thesequence of interest and primers dimers or non-specific amplification. It can alsobe used to detect different alleles. Non-specific detection detects all doublestranded DNA produced during the reaction.

Figure 2: The polymerase chain reaction. (K. Mullis and F. Faloona 1987). The red and pinklines represent the primers that the polymerase extends from. The blue and black lines representthe single stranded DNA template produced from denaturation of the double stranded DNAtemplate.

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SNON SPECIFIC DETECTION SYSTEMS

The standard method for non-specific detection is a double stranded DNAintercalating dye that fluoresces once bound to the DNA. The most commonly useddye is SYBR Green™ I. This dye is excited at 497 nm and emits at 520 nm. Theproducts detected by SYBR Green™ I can be observed by doing a melt curve at theend of the reaction. The reaction is heated slowly from 40 °C to 95 °C whilstcontinuously monitoring the fluorescence. The point at which the double strandedDNA melts is observed as a drop in fluorescence as the SYBR Green™ I dissociates.Different length products and products of different sequences will melt at differenttemperatures and be observed as distinct peaks when plotting the first negativederivative of fluorescence vs. temperature (many of the machines will do thiscalculation in the analysis software). If the PCR reaction is fully optimised it ispossible to produce a melting peak profile that contains only a single peak thatrepresents the specific product expected from the primer pair. In this situation SYBRGreen™ I can be useful for quantification and also for mutation detection. Evenamplicons that differ by a single nucleotide will melt at slightly different temperaturesand can be distinguished by their melting peaks. In this way it is possible todistinguish homozygotes (single peak) from heterozygotes (2 peaks). SYBR Green™I can often be useful for optimising a PCR reaction and checking that the primers areworking well before moving on to use one of the specific methods. An alternative toSYBR Green™ I is SYBR Gold™ (Molecular probes). The spectrum of this dye isslightly shifted compared with SYBR Green™ I, with the excitation peak at 495 nmand the emission peak at 537 nm. As most machines are set up for SYBR Green™I the SYBR Gold™ emission peak will be missed by the machines unless otherwisecalibrated. The advantage of using SYBR Gold™ is that it is far more stable thanSYBR Green™ I, which degrades very quickly.

The alternative to using a SYBR dye for non-specific detection is the Amplifluor™Universal Amplification and Detection System (Figure 2a). This system has 2 stepscarried out in the same PCR reaction. One of the specific primers has a universalsequence (Z sequence) added to the 5’ end. During amplification the Z sequence isincorporated into the amplicon. A second primer (Uniprimer™) that iscomplementary to the Z sequence is then used for amplification. The Uniprimer™has a hairpin structure attached to its 5’ end. The hairpin is similar in structure to aMolecular Beacon, as described below, but the single stranded loop sequence is notspecific to the amplicon sequence and does not bind to the amplicon. The hairpinis incorporated into the PCR product when the Uniprimer™ extends. When theextension product becomes a template for the opposite primer the taq extendsthrough the hairpin loop causing the loop to open. This separates the fluorophoreand quencher that were previously held in close proximity by the hairpin. Acorresponding increase in fluorescence is then observed.

SPECIFIC DETECTION SYSTEMS

There are a number of specific detection systems that use probes. These probescan be labelled with a range of dyes such as those mentioned in theinstrumentation section as well as others such as the Cy Dyes™ (e.g. Cy5™ andCy3.5™). Their different excitation and emission spectra allow the dyes to bedistinguished from one another. Alternative dyes that improve signal and makeprobe synthesis more efficient are also being developed. Below are some of themost popular probe methods currently used.

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Double-Dye Oligonucleotide probes

Double-Dye Oligonucleotide probes are the most widely used type of probe andare often the method of choice for scientists who have just started using Q&QPCR. They were developed by Roche and ABI from an assay that originally useda radiolabelled probe (Holland et al. 1991) and consist of a single strandedprobe sequence that is complementary to one of the strands of the amplicon. Afluorophore is attached to one end of the probe and a quencher to the other end.The fluorophore is excited by the machine and passes its energy, via FRET, to thequencher. Traditionally the FRET pair has been FAM as the fluorophore andTAMRA as the quencher. TAMRA is excited by the energy from the FAM and fluoresces. As this fluorescence is detected at a different wavelength to FAM thebackground level of FAM is low. The probe binds to the amplicon during eachannealing step of the PCR. When the taq extends from the primer bound to theamplicon it displaces the 5’ end of the probe which is then degraded by the 5’-3’ exonuclease activity of the taq. Cleavage continues until the remainingprobe melts off the amplicon. This process releases the fluorophore andquencher into solution, spatially separating them compared to when they wereheld together by the probe (Figure 3). This leads to an irreversible increase influorescence from the FAM and a decrease in the TAMRA.

Double-Dye Oligonucleotide probes can be used for qualitative, quantitativeapplications and mutation detection and most designs appear to work well. Formutation detection the probe is designed to hybridise over the mutation and canbe made specific enough to detect single base differences. To obtain robustallelic data it is vital that a probe is used for each different mutation otherwisenegative results with one probe, caused by failed PCR reactions, could be mis-scored as an absence of a particular allele. Ideally these probes would bemultiplexed to make the assays faster and cheaper.

TAMRA, whilst being used as a quencher, can also be used as a fluorophore.

Figure 2a: Amplifluor mode of action

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Using a different quencher for Double-Dye Oligonucleotide probes would free upTAMRA to improve multiplexing capability. There are now several darkquenchers available. These absorb the energy emitted by the fluorophore butrelease it as heat rather than fluorescence. Examples of these are methyl red orDABCYL (Nasarabadi et al. 1999) and the new ElleQuencher™ and Eclipse™Dark Quencher. Methyl red quenches the lower wavelength dyes such as FAMbut is not good at quenching those that emit at a higher wavelength, e.g. Cy5™.ElleQuencher™ was designed to quench the higher end of the spectrum. It hasbeen tested in Double-Dye Oligonucleotide probes and Scorpions™. It givesgood results for both when tested with dyes such as ROX and TAMRA but is alsoequivalent or better than methyl red for dyes such as FAM (lower wavelength).For further details see www.eurogentec.com or www.oswel.com.

Multiplexing is often a question difficult to address and the choice of the correctfluorophore dyes combination is therefore crucial. Eurogentec is a licensedprovider of a new fluorescent dye developed by Epoch Biosciences. This newfluorophore-phosphoramidite, called Yakima Yellow™ from the name of the

Figure 3: Double-Dye Oligonucleotide‚ mode of action

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S Yellow Sally of the Yakima river, can conveniently be incorporated under normalfluorescent probe synthesis conditions. Use of the Yakima Yellow™ dyesignificantly increases both the number of independent reporters possible in anassay, and thus increases the wavelength range available for use. For bestresults, the combination of FAM (target) and Yakima Yellow™ (endogenouscontrol) is recommended (they have the largest difference in emission maximum)whereas JOE and Yakima Yellow™ should not be combined. As so, YakimaYellow™ offers the most affordable alternative to the use of the VIC™ dye fromABI (absorption and emission maximum wavelengths are respectively of 528 nm – 546 nm for VIC™ and 525 – 548 nm for Yakima Yellow™).

Eclipse™ probes

A new probe system that uses the minor groove binder (MGB) technology isEclipse™. These are short linear probes that have the minor groove binder andquencher on the 5’ end and fluorophore on the 3’ end. This is the oppositeorientation to Double-Dye Oligonucleotide probes and it is thought that the minorgroove binder prevents the exonuclease activity of the taq from cleaving theprobe. Quenching occurs when the random coiling of the probe in the free formbrings the quencher and fluorophore close to one another. The probe isstraightened out when bound to its target and quenching is decreased, leadingto an increase in fluorescent signal. The minor groove binder works together withthe quencher to improve quenching, reducing the background level offluorescence. As with minor groove binder Double-Dye Oligonucleotide probes,short probes can be used and allelic discrimination is good.

Sensitivity tests have shown that the probes can detect down to a few copies,when asymmetric PCR is used, and that amplification is linear over a wide rangeof concentrations. These properties make Eclipse™ probes suitable forquantitative and qualitative PCR.

Figure 4: Eclipse™ probes

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SMolecular Beacon probes

Molecular Beacons (Tyagi and Kramer 1996) differ from Double-DyeOligonucleotide probes in both their structure and mode of action. They consistof a hairpin loop structure where the loop is a single stranded probe that iscomplementary to the amplicon. The stem is approximately 6 bases long, shouldmainly consists of C’s and G’s and holds the probe in the hairpin configuration.A fluorophore is attached to one end of the stem and a quencher (usually methylred or DABCYL) to the other. The stem holds the two in close proximity andquenching occurs by collisional quenching (Figure 5).

When the amplicon is produced during PCR, the probe is able to bind to aspecific sequence providing the design is such that the probe-target duplex isthermodynamically more stable than the hairpin structure at the fluorescentacquisition temperature. Once the probe binds to its target the hairpin is openedout and the fluorophore and quencher are separated. The increase influorescence that occurs is reversible, unlike Double-Dye Oligonucleotide probes,as the probe will dissociate at high temperatures and close back into the hairpinstructure. Eventually, as temperature is increased, the stem will melt giving alinear probe that is high in fluorescence. This gives a melt curve that is very usefulfor observing the dynamics of the reaction and the best temperature forfluorescent acquisition (Figure 6).

The stem structure adds specificity to this type of probe as the hybrid formedbetween the probe and target has to be stronger than the intramolecular stemassociation. Mismatched targets will form a probe-target duplex but this willdissociate at a lower temperature than a perfectly matched duplex. Theassociated differences in the melt curves will show this and a mutation detectionassay can be developed to monitor fluorescence at a temperature where theprobe binds to the perfectly matched target but has dissociated from amismatched target. One problem with Beacons is that although good designscan give nice results, often the signal is very poor.

Figure 5: Molecular Beacon probes

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An easy way to improve signal strength is to use wavelength-shifting MolecularBeacons (Tyagi et al., 2000). These Beacons are nucleic acid probes thatcontain a harvester fluorophore that absorbs strongly in the wavelength range ofthe monochromatic light source, an emitter fluorophore of the desired emissioncolor, and a non-fluorescent quencher. In the absence of complementary nucleicacid targets, the probes are non-fluorescent, whereas in the presence of targets,they fluoresce, not in the emission range of the harvester fluorophore thatabsorbs the light, but rather in the emission range of the emitter fluorophore. Thisshift in emission spectrum is due to the transfer of the absorbed energy from theharvester fluorophore to the emitter fluorophore by FRET, and it only takes placein probes that are bound to targets. Wavelength-shifting Molecular Beacons aresubstantially brighter than conventional Molecular Beacons that contain afluorophore that cannot efficiently absorb energy from the availablemonochromatic light source. Eurogentec proposes these new Molecular Beaconprobes either quenched with DABSYL or with our new ElleQuencherTM.

During the past few years, Eurogentec has gained substantial experience in theuse and design of Molecular Beacons. This was achieved through collaborativeworks, especially with the Department of Molecular Cell Biology in Leiden(Molenaar et al., 2001 ; Szuhai et al., 2001 (1) ; Szuhai et al., 2001 (2).

Scorpions™ probes

Scorpions™ differ (Whitcombe et al. 1999; Thelwell et al. 2000) from thespecific detection methods discussed so far in that their mechanism of probing isintramolecular. Their structure is as follows: the hairpin loop is linked to the 5’end of a primer via a PCR stopper. After extension of the primer during PCR

Figure 6: Melt curve of a Molecular Beacon with and without a synthetic complement as a target. Annotations show the configuration of the Beacon and target at different temperatures.

With synthetic targetNo target

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Samplification, the specific probe sequence is able to bind to its complementwithin the same strand of DNA. This hybridisation event opens the hairpin loopso that fluorescence is no longer quenched and an increase in signal is observed(Figure 7).

The PCR stopper prevents read-through, which could lead to opening of the hairpinloop in the absence of the specific target sequence. Such read-through would lead tothe detection of non-specific PCR products, e.g. primer dimers or mispriming events.

Unimolecular probing is kinetically favourable and highly efficient. Covalentattachment of the probe to the target amplicon ensures that each probe has a targetin the near vicinity. Enzymatic cleavage is not required, thereby reducing the timeneeded for signalling compared to Double-Dye Oligonucleotide probes, which mustbind and be cleaved before an increase in fluorescence is observed.

Scorpions™ primers have successfully been used for mutation detection andquantification, having the specificity and melt curve analysis of the MolecularBeacons with additional speed and efficiency. Duplex Scorpions™ (Figure 8) havealso been developed to improve signal intensity (Solinas et al. 2001). In standardScorpions™ the quencher and fluorophore remain within the same strand of DNAand some quenching can occur even in the open form. In Duplex Scorpions™ thequencher is on a different oligonucleotide and separation between the quencherand fluorophore is greatly increased, decreasing the quenching when the probe isbound to the target. Eurogentec is a licensed supplier of Scorpions™ probes.

Figure 7: Scorpion unimolecular mode of action. Progression through a PCR reaction

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Hybridization probes

Roche has developed hybridization probes (Caplin et al. 1999) for use with theircapillary system. Two probes are designed to bind adjacent to one another on theamplicon. One has a 3’ label of FAM, whilst the other has a 5’ dye, LC red 640 or705 (alternative dyes for hybridization probes are ROX for channel 2 and Cy5™for channel 3, all available from Eurogentec; www.eurogentec.com). During thePCR reaction, the 2 probes bind to the specific amplicon sequence and the capillarysystem excites the FAM. This passes its energy on to the dye through FRET and theincrease in fluorescence, proportional to the increase in amplicon, can be observedin channels 2 or 3 depending on the dye used. This technology can be used forquantitative and qualitative PCR or mutation detection. For mutation detection oneprobe is positioned over the polymorphic site. Melt curve analysis after the PCRindicates which alleles are present since the mismatch causes the probe to dissociateat a different temperature to the fully complementary amplicon. One probedissociating from the amplicon causes a decrease in fluorescence as FRET can nolonger occur. It should be noted that FRET is not efficient between dyes and FAM,particularly the LC red 705 dye, due to the small overlap between the spectra.

Figure 8: Duplex Scorpions™ mode of action. Key is as for Figure 7

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SOTHER TECHNOLOGIES

The technologies that have been discussed above are the most widely usedtoday but there are new technologies being developed, e.g. Hy-Beacon™probes.

ResonSense® probes

ResonSense® probes are single labelled probes that bind to a specific template.A DNA intercalater, such as SYBR Gold™, which can pass energy to the dye onthe probe via FRET, is also added to the reaction. Once the probe has bound toits target it gives a double stranded template for the SYBR Gold™ to bind to.Energy is passed from the SYBR Gold™ (excited by the machine) to the dye onthe probe leading to an increase in fluorescence from the dye. This technologyworks for both quantitative PCR and allelic discrimination.

Light-up probes

Light-up probes (Wolffs et al. 2001 - Svanvik et al. 2000) are anotheralternative, which use the ability of some dyes to bind to the target and increasein fluorescence once the probe has bound.

These probes are PNA (Peptide Nucleic Acids) rather than DNA. PNA hasproperties that make the hybridisation faster and stronger than when using DNAprobes.

Figure 9: ResonSense® Technology. Green circles represent SYBR Gold™, yellow circles represent the dye used to label the probe. Red lines are primers. The green line is the probe.Red lines with arrows indicate energy transfer, whilst the turquoise line with an arrow indicatesexcitation by the Q&Q PCR machine. The purple lines indicate the extension products from theprimers.

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S Hy-Beacon™ probes

Hy-Beacon™ probes have been developed by the LGC (Laboratory of theGovernment Chemist) and consist of a specific linear probe with a dye label.Assays use the change in fluorescence observed when the probe binds to itstarget, compared to when it is in its single stranded form, for sequence detection.

QUENCHING AND FRET

Quenchers are molecules that can accept energy from a fluorophore anddissipate the energy by one of two mechanisms, proximal or FRET quenching

A fluorophore absorbs light energy and is promoted to an excited state, in theabsence of a quencher the fluorophore falls back to the ground state and theexcess energy is released as fluorescence.

Proximal quenching

When the fluorophore is in close proximity of a quencher molecule the energy istransferred from the fluorophore to the quencher, which then dissipates theenergy as heat and therefore no fluorescence is observed. This is sometimesknown as collisional quenching.

FRET quenching

The fluorophore transfers its energy to the quencher (which may be anotherfluorophore), and the energy is released from the quencher as radiative decay(i.e. fluorescence) at a higher wavelength. The efficiency of the process isdependent on 1/r6 (Förster distance) where r is the fluorophore - quencherdistance.

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Examples

Molecules that can quench fluorescence are increasingly important in moleculargenetics.

Many of the commonly employed techniques for the detection of nucleic acidsequences in a homogeneous manner use fluorescence as the signalingtechnology. Typically a single stranded probe is labelled with a fluorophore andquencher molecule. Changes in quenching of the fluorophore caused byhybridisation of the probe to its target nucleic acid leads to signal generation.

Conformational probe

Examples of such methods involving conformational probe changes to separatethe fluorophore and quencher are Scorpions™ primers and Molecular Beacons.In these systems fluorescence quenching is proximal due to the close contact ofthe fluorophore and quencher.

Cleavage

Double-Dye Oligonucleotide probes are an example of probes that requireenzymatic cleavage to separate the fluorophore and quencher. Here, themechanism is FRET quenching. The fluorophore donor transfers its energy to thequencher acceptor, which releases the energy as light of a higher wavelength.Close contact of the fluorophore and quencher is not required.

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S Classical quenchers

Proximal quenchers DABCYL and Methyl Red, can be used to quench dyes suchas FAM, TET and TAMRA in Molecular Beacons and Scorpions™ Primers. Otherquenchers are suitable for Double-Dye Oligonucleotide probes, such as TAMRA,DABCYL, Methyl Red and our new ElleQuencher™ and Eclipse™ DarkQuencher.

NEW QUENCHERS

The ElleQuencher™

The ElleQuencher™ is a proprietary molecule that quenches a wide range ofdyes, absorbs at the longer wavelengths of the visible spectrum, is completelynon-fluorescent and is an extremely low background quencher. ElleQuencher™significantly improves the signal/noise ratio for a large selection of fluorescent dyes.This quencher is for use in Real-Time, quantitative PCR, SNP detection, allelicdiscrimination, spectral Genotyping and in situ hybridisation and is suitable forDouble-Dye Oligonucleotide, Scorpions™, Molecular Beacons or wavelength-shifting Molecular Beacon probes.

Figure 11: UV-vis absorbance spectra of DABCYL (A), Eclipse™ Dark Quencher (B) andElleQuencher™ (C)

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SOther available dye/quencher pairs like FAM - TAMRA suffer from a number ofdrawbacks; these include intrinsic fluorescence of the quencher and poorspectral overlap between the fluorescent dye and quencher molecule, bothresulting in a poor signal-to-noise ratio. It is known that the currently available"universal" quenchers do not significantly reduce the fluorescence of severalcommonly used fluorophores.

ElleQuencher™ (EQ) is non-fluorescent thus eliminating any complications ofbackground signals that can arise when fluorophores are used as quenchers. Itabsorbs over a large section of the visible spectrum, and therefore efficientlyquenches most of the commonly used fluorophores, especially those emitting athigher wavelengths.

The absorption spectra of the ElleQuencher™ and Methyl Red are shown inFigure 11. Generally the efficiency of energy transfer from a fluorophore toquencher, increases proportionally with the degree of spectral overlap betweenthe emission of the fluorophore and the absorption of the quencher. Theabsorption spectra of the ElleQuencher™ shows that the ElleQuencher™ willquench lower wavelength dyes (e.g. FAM) as efficiently as Methyl Red but willbe far superior for the longer wavelength dyes (e.g. Cy5™)

The Eclipse™ Dark Quencher

Along with the ElleQuencher™ (EQ), Eurogentec proposes the Eclipse™ DarkQuencher (DQ). This new non-fluorescent molecule quenches effectively over abroader wavelength range from about 400-650 nm in contrast with DABCYL(360-560 nm). The Eclipse™ Dark Quencher is useful in any detection systemthat involves FRET (i.e. in Beacons). It is particularly suitable in combination withFAM and Yakima Yellow™ for multiplexing (Figure 11).

Efficient quenching of fluorophores

When the hybridisation probes are annealed, the fluorophore and quencher areheld in close proximity and the duplex is non-fluorescent. Heating the duplexcauses the strands to dissociate and the change in fluorescence can bemeasured. The efficiency of the quenching can be calculated by the signal/noiseratio (Figure 12).

Figure 13 shows that the ElleQuencher™ is a superior quencher for TAMRA,Cy3™ or Cy5™ and gives comparable quenching with FAM. The four dyeschosen represent a broad range of wavelength emission maxima: FAM (520 nm), Cy3™ (570nm), TAMRA (580 nm) and Cy5™ (670 nm).

Figure 12: Schematic representation of an experimental procedure designed to compare theefficiency of different quenchers

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Figure 13: Comparative analysis of quenching efficiencies of 3’-ElleQuencher™ (EQ) and 3’-Methyl Red (MR) for 4 different dyes

Methyl Red dU

N N

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DABCYL466 nm abs 453 nm absNo emission No emission

6-TAMRA565 nm abs

580 nm emission

Methyl Red410 nm absNo emission

CHEMICAL STRUCTURE OF THE MOST COMMON QUENCHERS

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SDYES AND QUENCHERS CHEMISTRY

There are a number of different dyes and quenchers available for use in qualitativeand quantitative PCR applications, the most common ones are listed in the table below.

Available Dyes Absorption (nm) Emission (nm)FAM 492 515TET 521 536JOE 527 548HEX 535 556TAMRA 555 580ROX 575 602Cy3™ 552 565Cy3.5™ 581 596Cy5™ 651 674Cy5.5™ 675 694Cy7 743 767R6G 518 543

Other dyesTexas Red™ 583 603VIC™ 528 546Yakima Yellow™ 525 548

QuenchersMethyl Red 410 N/AElleQuencher™ 650, 600 N/ADABCYL 453 N/ADABSYL 466 N/ATAMRA 555 580Eclipse™ Dark Quencher 500 N/A

There are several methods by which a dye can be incorporated into a probe.The most efficient of which is via a phosphoramidite or at the 3’-end using a dyelabeled solid support, since the dye is added during solid phase DNA synthesis.The most common general structures are shown below.

Those attached on the C6 linker (1) are usually added to the 5’-end of the oligobut combined with the use our branched monomers (5,6), these can beincorporated within the sequence. The use of the branched monomers alsoallows multiple dye additions with an oligonucleotide and although they areshown with a base labile protected branch, these are also available withphotolabile protecting groups. This allows there use with additionalmodifications normally unstable to base.

There are three structures used to incorporate internal or 3’ labels, 1,2-diol type(2), dRibose type (3) and 5-X-dU (4). These all work well but the most efficientare the latter two since these do not disrupt the normal phosphate sugarbackbone and are less susceptible to side reactions. There is potential for areaction to occur between the phosphate backbone and the free hydroxyl groupof the 1,2- (or 1,3-diol) when incorporated at the 5’-end of the oligonucleotideresulting in the loss of the reporter group.

Not all dyes are available as phosphoramidites or solid supports and these have

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S to be incorporated post synthetically using dye-active esters (e.g.rhodaminedyes). This is the least efficient method of labeling since the coupling reaction iscarried out in solution and is low yielding in comparison with solid phasesynthesis. The active ester is attached to the oligo via an amino modifiedphosphoramidite which have the same general structures as those shown below.

General structures of dye labeled oligos

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Branching monomers

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S ApplicationsQUANTITATIVE PCR

Quantitative PCR (for reviews see Bustin 2000 - Freeman et al. - Halford 1999 -Heid et al. 1996) can be used for a variety of applications such as monitoringgene expression levels and viral load. Monitoring Gene expression is one of themost popular applications for Real-Time quantitative PCR, being used forassessing the effects of different treatments on the level of mRNA transcription.As mRNA is being measured the experiment must contain a reverse transcriptionstep where RNA is converted to cDNA. cDNA differs from genomic DNA bycontaining only DNA complementary to the mRNA, i.e. it does not contain anyintrons. Reverse transcription can be a separate step carried out before thequantitative Real-Time PCR (2 steps) or can be combined with this process (1step). As with PCR it has to contain primers. It is possible to use the specificprimers required for the PCR step and these are always used in a 1 stepquantitative Real-Time PCR. Note that the RT step is a linear process rather thanexponential and it is the reverse primer that is used to produce the cDNA strandof the mRNA. In a 2 step reaction there is a choice of assay specific primers,random hexamers or nonamers or oligo dT. If the target is rRNA oligo dT maynot be used as this primes from the poly A tail found on mRNA. The merits of thedifferent primers for mRNA is a much discussed subject, with some thinking thatusing specific primers will introduce a bias and others feeling that using oligodT, priming at the 3’ end of the transcript will reduce the representation of 5’regions of the transcripts. It has also been suggested that a mixture of oligo dTand random hexamers should be used. Once the cDNA has been synthesised astandard quantitative Real-Time PCR reaction can be run and quantificationcarried out by the machine’s software.

Quantitative PCR software uses the exponential phase of PCR for quantification.PCR is initially an exponential process but eventually reaches a plateau phasecaused by one of the reagents becoming limiting. The fluorescent signal detectedat the plateau phase, with baseline subtraction, is called the Rn value. Anothervalue, called ∆Rn, is often used when a passive reference (see section onprotocols) is included in the reaction. ∆Rn is the Rn value normalised against thepassive reference signal. Reactions can plateau at different levels even if theyhave the same starting concentration of target. During the exponential phase theamount of target should be doubling every cycle and there will be no bias dueto limiting reagents. Analyses use the Ct, the point (cycle number) at which thesignal is detected above the background and is in the exponential phase. Themore abundant the template the earlier this point is reached. Differences in Ctthen have to be related to some other quantitative value to make themmeaningful. There are 2 types of quantification:

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SRelative quantification

Relative quantification is the most widely used technique. An endogenous controlis amplified from the sample as well as the gene of interest. Relative geneexpression comparisons work best when the gene expression of the chosenendogenous control is more abundant and remains constant, in proportion tototal RNA, among the samples. By using an endogenous control as an activereference, quantitation of a mRNA target can be normalized for differences inthe amount of total RNA added to each reaction. For this purpose, the mostcommon choices are 18S rRNA, GAPDH (glyceraldehyde-3-phosphatedehydrogenase) and ß-actin mRNA. Because the 18S rRNA does not have apoly-A tail, cDNA synthesis using oligo-dT should not be used if 18S RNA willbe used as a normalizer. The issue of the choice of a normalizer has recentlybeen reviewed by Suzuki (2000). The authors recommend caution in the use ofGAPDH as a normalizer as it has been shown that its expression may beupregulated in proliferating cells. They recommend ß-actin as a better activereference. GAPDH is also severely criticized as a normalizer in another recentreview (Bustin 2000). Caution should also be exercised when 18S rRNA is usedas a normalizer as it is a ribosomal RNA species (not mRNA) and may notalways represent the overall cellular mRNA population. Since the chosen RNAspecies should be proportional to the amount of input RNA, it may be best to usea combination as normalizer. It is therefore desirable to validate the chosennormalizer(s) for each target cell or tissue studied. It should be expressed at aconstant level at different time points by the same individual and also by differentindividuals at the target cell or tissue (for example, peripheral bloodlymphocytes). Normalisation of the experimental data to this gene removeserrors caused by slight variation in PCR efficiencies between samples anddifferent amounts of template. There are 2 ways of using an endogenous control.The first is to run a standard curve of known amounts (in units, the absoluteamount is not required) of both the endogenous control and gene of interest. Theratio of specific gene signal to endogenous control signal in a sample can thenbe calculated in each sample. One sample has to be nominated as thecalibrator, e.g. the untreated sample. Each normalised value can then be dividedby the calibrator to get relative values for each sample. A second way of usingthe endogenous control is to compare Ct values without running a standardcurve. The following equations are used:

∆Ct= endogenous Ct-Gene of interest Ct

∆∆Ct= ∆Ct of sample-∆Ct of calibrator

Amount of target normalised to a control and relative to a calibrator = 2(∆∆Ct)

It is important when using this method that the PCR efficiencies of the 2 reactionsare similar. If the efficiencies are similar the ∆Ct should not change with differentamounts of total sample added. Ideally both should have a standard curve slopeof approximately –3.3 for 100 % efficiency.

The endogenous control can be used as a single-plex reaction where the geneof interest and control are amplified in separate reactions. It can also be used ina multiplex reaction, which prevents pipetting errors between the control reactionand gene of interest reaction that can occur when using single-plex reactions.Multiplexing quantitative Real-Time PCR reactions is possible as the machines

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S can detect more than one dye in each sample. It is important to consider whichdyes are best for multiplexing on the particular machine being used and also anyrequirements for spectral calibration (see our new Double-Dye Oligonucleotidescombinations including 5’ FAM and 5’ Yakima Yellow™). This is requiredbecause dyes exhibit some spectral overlap, making it necessary to calibrate themachine to enable it to distinguish between the dyes. Spectral calibration kitscan be bought for the majority of machines where this needs to be considered.Problems arise when the endogenous control and gene of interest compete forPCR reagents. A highly abundant endogenous control can out-compete a lessabundant template leading to a bias in the results. Limiting the primerconcentration for the most abundant template can avoid this competition.However, this takes a considerable amount of development. Primerconcentrations that do not alter Ct compared to a fully optimised reaction butlead to a much earlier plateau phase need to be chosen. This means that thereagents are not used up and competition is no longer a factor.

Absolute quantification

Absolute quantification requires a standard curve of known quantities to give aprecise value for copy number per cell, total RNA or unit mass of tissue. Thisquantification requires a standard curve of known copy numbers. It can beconstructed using several methods. The amplicon being studied can be cloned,or a synthetic oligonucleotide used. Either DNA or RNA standards can be used.For RNA standards the cloned amplicon is transcribed to RNA or a singlestranded sense oligonucleotide made. There are several criteria for absolutestandards. The standard must be amplified using the same primers as the geneof interest and must amplify with the same efficiency. The standards must also bequantified accurately. This can be carried out by reading the absorbance atA260, although this does not distinguish between DNA and RNA, or by usinga fluorescent nucleic acid stain such as RiboGreen®. Once a copy number valuehas been obtained for a sample it must be normalised to something. Oftennumber of cells or total RNA is used. This requires accurate measurement of inputRNA, again either by reading the absorbance or using a nucleic acid stain.Alternatively, an exogenous control can be used for normalisation.

QUALITATIVE PCR

Among qualitative PCR applications, allelic discrimination becomes more andmore important. This method can be used for a number of applications such asdisease gene detection (e.g. CFTR, Thelwell et al. 2000). When using SYBRGreen™ I, alleles can be distinguished by the difference in amplicon meltingpeaks caused by the slight differences in sequence. Specific detection methodsare more often used for these applications and probes can be used to detectsingle base mutations or small deletions. They can also be used to detectdifferent splice sites. The design and optimisation of the assays for allelicdiscrimination is vital and is discussed further in later sections. An alternative toplacing the probe over a polymorphism is to place the 3’ end of one of theprimers over the mutation. A mismatch in this region of the primer should preventamplification and the probe can be used to simply detect whether there isspecific amplification or not. It is often the case that mismatched primers willamplify the template to some degree but a difference in Ct will be observed.

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designINTRODUCTION

When designing the primers and probes for a Real-Time assay the firstconsideration is whether there are any restrictions on the designs. For example,if the assay is to be used to study gene expression levels it is a good idea todesign the primers in adjacent exons and the probe across the exon/exonboundary. The assay will then not pick up any DNA contamination of the RNAtemplate, unless that particular gene does not contain any introns. For mutationdetection the amplicon must be designed around the mutation site with oneprimer close to the mutation. As discussed earlier it is possible to get goodmutation detection by placing the 3’ base of a primer over the mutation site. Thisobviously places constraints on the positioning of the primer. If the probe isdesigned to detect a mutation, rather than the primer, the mismatch created willdetermine the length of probe. For example, G-T mismatches are very stable andthe probe will need to be fairly short to make the mismatch significant enough toprevent the probe binding. The sequence surrounding the mutation will alsoaffect the stability of a mismatch.

For non-specific and specific detection methods primers can be designed usingsoftware such as Primer Express™ (ABI) or Oligo™ (Molecular Biology Insights,Inc). These can be used to obtain the correct Tm’s and check for secondarystructure and primer dimers. The Tm’s of the 2 primers should be similar and theyshould have as little secondary structure as possible. Both programs will giveoptimal annealing temperatures for the reaction. It is important to keep theamplicons short, between 50 and 150 bases. This allows the probe sequencesto successfully compete with the complementary strand of the amplicon andmakes the reaction fast and efficient. However, longer amplicons have beenused successfully. It is also important to check for primer dimers that can formbetween sets of primers that are to be multiplexed together. Multiplexed primerpairs must all work well at the same annealing temperature. If the software forprimer design is not available it is possible to design primers by eye. Choseregions that are unlikely to cause secondary structure problems that are 20-80% GC. The Tm can be calculated using the rule of 2 °C for an A or T and 4 °Cfor a G or C. Most primers are around 20 bases in length but can be between9 and 40 bases. It is advisable to keep primers 17 bases or longer as below thisthe chance of finding a random primer binding site becomes more significant.Runs of G’s and C’s should be avoided at the 3’ end, with a maximum of 2 G/Cbases in the final 5 bases.

DOUBLE-DYE OLIGONUCLEOTIDE DESIGN

For the specific detection methods both primers and probes need to be designed.The design is dependent on the technology chosen. The simplest way of

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IGN designing a Double-Dye Oligonucleotide probe is to use Primer Express™. This

software was contains all the parameters for designing Double-DyeOligonucleotide probes and minor groove binder Double-Dye Oligonucleotideprobes. Exon/exon boundaries can be highlighted and parameters for mutationdetection can be entered. As all probes are designed under the same parametersmost probes should be suitable for multiplexing together. If Primer Express™ isnot available probes can be designed by eye. Most common design rulessuggest that the 5’ end of the probe should be as close as possible to the 3’ endof the primer. The Tm of the probe should be 10 °C higher than the primer Tmunless it is being used for mutation detection, in which case the Tm differenceshould be 7 °C. This destabilises the probe so that when the 5’ end is displacedby the taq the mismatch should cause the remainder of the probe to dissociatebefore cleavage can occur. The probe should be 9-40 bases, 20-80 % GC’s,have less that 4 contiguous G’s and no more G’s than C’s. There must not be aG on the 5’ end as this can quench the fluorophore.

SCORPIONS™ DESIGN

When designing Scorpions™ the folding of the primer and probe is the crucialfeature. The probe sequence should be close to the 3' end of the primer youintend to attach the probe to. The probe should be complementary to theextension of this primer, making probing intramolecular. In order to be in thecorrect orientation the probe should be written so that the 5' end iscomplimentary to the 3' end of the target, i.e. the probe should be the reversecomplement of the target. Probe sequences should be about 17-27 bases. Theprobe target should be 11 bases or less from the 3' end of Scorpions™.Although a greater distance works, the further away the probe target the lowerthe probing efficiency and the advantage of using an intramolecular probe islost. A stem sequence of 6 or 7 bases, mostly C’s and G's, avoiding repetitivemotifs e.g. CGCGCG, should be added to either end of the probe so that the 2regions of stem can bind to one another and hold the probe in a loopconfiguration. The 5' stem sequence should begin with a C as a G may quenchthe dye.

The following website: http://bioinfo.math.rpi.edu/~mfold/dna/form1.cgishould be used to fold Scorpions™ (probe, stem and primer) at 0.004 M Mgand 0.01 M Na. The folding temperature should be the temperature at whichthe fluorescence will be monitored. This parameter often needs optimising and agood starting temperature for the folding is 60 °C. Fold both the Scorpions™sequence alone and the Scorpions™ with amplicon. The design should be suchthat a stem loop is produced for Scorpions™ alone and the probe is bound tothe target for Scorpions™ with amplicon. This may mean altering the stemsequence and/or length of the probe sequence or adjusting the temperature.The ∆G value should be negative and ideally the stem loop should have a Tm of5-10 °C higher than the probe bound to the target. The ∆G value is moreimportant. When designing a probe for mutation detection the same principlesapply. A design should be found where the probe is bound to the perfectlymatched target but has dissociated from the target with a mismatch. Once thesequence has been designed the fluorophore is placed on the 5’ end and amethyl red quencher and hexaethylene glycol molecule (PCR stopper) placedbetween the primer and the hairpin loop. If Scorpions™ are being used on thecapillary systems for detection on channel 2 (640 nm) a specific design is

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IGNrequired. As the capillary system will only excite at 470 nm (FAM) it is necessary

to incorporate a FAM into Scorpions™. This is placed within the stem, attachedto a T. Consequently the stem must have an AT base pair within it. A ROX isplaced on the 5’ end of the oligonucleotide (Figure 14).

During the PCR the FAM is excited and passes its energy on to the ROX. This isquenched in the closed form of the Scorpions™ but once the probe binds to theamplicon the increase in ROX fluorescence can be observed. The FRET betweenthe ROX and FAM is extremely good and the FAM signal observed in channel 1remains constant so that a FAM Scorpions™ can be multiplexed with the FRETScorpions™ without interference from the ROX. However, the FAM signal willshow up in channel 2 and it is necessary to remove this crosstalk by using theRoche colour compensation kit.

MOLECULAR BEACON DESIGN

Design of Molecular Beacon hairpin loops is fairly similar to Scorpions™ designexcept that intramolecular folding does not need to be taken into account. Thehairpin structure needs to be checked in the DNA folding programme. The Tmof the Beacon probe sequence should be 7-10 °C higher than the annealingtemperature of the primers. In contrast to the probes discussed above MolecularBeacons should be designed in the centre of the amplicon with greater than 6bases between the Beacon and 3’ ends of the primers.

HYBRIDIZATION PROBE DESIGN

Hybridization probes should be designed so that the Tm’s of the 2 probes arewithin 2 °C of each other, unless performing mutation detection, and 5-10 °Chigher than the primer Tm. Mutation detection probes should have a differencein Tm of 5-10 °C and the more unstable probe should be sited over the mutation.As with other probes secondary structure should be avoided where possible. The2 probes should be adjacent to one another on the amplicon with a spacing of1-5 bases between them. The probes tend to be 23-35 bases long and the 5’labelled probe should have a PCR blocker on the 3’ end to prevent it beingextended by the taq (Landt and Nitsche).

Once the sequence has been designed the dyes and quenchers should bechosen so that the machine of choice can detect the probes.

Figure 14: ROX-FAM FRET Scorpions™ detected in channel 2 of the Roche capillary system.

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There are now a number of kits available for Q&Q PCR that have a simpleprotocol for the user to follow. Standard cycling conditions (ABI) for Double-DyeOligonucleotide‚ are as follows

50 °C for 2 minutes95 °C for 10 minutes40 cycles of95 °C for 15 sec60 °C for 1 minute

This is a 2 step PCR where the extension and annealing step are a combined 60 °Cstep. This is possible since the amplicons are designed to be very short and canbe copied without requiring a 72 °C extension. This protocol should be usedinitially when testing a new assay.

Double-Dye Oligonucleotide probes are designed by Primer Express™ to workoptimally under these conditions. However, Molecular Beacons and Scorpions™may need an extra step for monitoring the fluorescence as the optimaltemperature for fluorescent signal or allelic discrimination may be different to theannealing/extension step. Melt curves are useful for determining thistemperature. The annealing temperature may require optimisation for any of thetechnologies.

In addition to the cycling parameters there are 2 temperature steps at thebeginning of the reaction that have particular functions. The 50 °C step is toallow UNG (uracil-N-glycosilase) to act. UNG is used as a contamination controlsystem. dUTP is used instead of dTTP (replacing at least a proportion of the dTTP)in most Q&Q PCR kits. UNG will weaken glycosidic bonds in DNA with U’sinstead of T’s. A heating step will then deactivate the UNG and break theglycosidic bonds fragmenting the DNA so it cannot be amplified. UNG is usefulif any PCR products from a previous amplification are contaminating the reactionat the start of the reaction. As one of the advantages of Q&Q PCR is that thereaction and analysis can be carried out without opening a tube, this type ofcontamination should not occur. The 95 °C for 10 minutes step is used to activatea hot start taq polymerase. A hot start enzyme is one that is inactive at lowtemperatures, e.g. room temperature at which the reaction is set up. Thisprevents non-specific amplification occurring before the actual amplification andcan reduce primer dimer formation. Heating to 95 °C activates the enzyme andamplification can begin once the primers are annealed. The annealingtemperature is reached by decreasing the temperature from 95 °C. This meansthat the most specific annealing temperature for the primers is reached first andthe first cycle should produce the specific product rather than amplifying anynon-specific DNA produced earlier. A 10-minute hold at 95 °C is required for ahot start enzyme that has a chemical modification. This time allows all theenzyme to be activated. It is also possible to use an antibody (e.g. ClontechTaqStart™) that inactivates the taq. This drops off the taq at 95 °C and does notrequire such a long hold.

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LS The reaction mix (based on a solution of Tris) is often specific to the machinebeing used, as is the total volume of the PCR. The PCR buffer is dependent uponwhether the reaction tubes are plastic or glass (capillary system). Glass chelatesreagents like taq, DNA and magnesium. Adapted mixes contain non-acetylatedbovine serum albumin (BSA) to prevent some of this chelation. Alternatively BSAat 250 ng/ml can be added to a homemade mix. pH can also be important butmost kits and buffers are optimised for their particular application. One otherrequirement for the plastic tubes is in mixes for the 7700. This machine uses aROX passive reference that remains constant throughout the reaction tonormalise the data for slight differences between wells, e.g. changes in volume.This should be mixed with the reaction buffer in the kits. The reaction buffer canbe bought as a 10x mix with the following reagents to be added to the mastermix; dNTP’s (normally at 200 mM), taq, magnesium chloride and sterile water.Alternatively these can be bought as a ready mixed 2x kit, which often containsglycerol as a stabiliser. Magnesium chloride is a component required by theenzyme that can affect specificity. The concentration of this should be optimisedbut is generally between about 4-7mM for Real-Time reactions. The concentrationof primers and probes should also be optimised to give the maximum signal withlowest Ct, whilst keeping the concentration of probe as low as possible todecrease cost. If the primers have the same Tm they should be balanced with thesame numbers of primer molecules binding at the annealing temperature.However, the theoretical Tm is not normally identical to the actual Tm. Slightlydifferent primer concentrations can be used to compensate for this and giveequal numbers of bound forward and reverse primers.

The protocol for the reverse transcription step also has to be considered. This willdepend upon whether the RT step is being carried out separately or as a singlestep reaction with the PCR. Reverse transcription is carried out by incubation ata constant temperature depending on the enzyme being used. Reversetranscription kits are available with optimised buffers and simple protocols.

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NSFrequently asked

questions1. Reaction that has worked well with a specificprobe system is no longer working.

The probe may have bleached if it has been left in the light for some time.Although the reaction is working the fluorophore is no longer reporting the result.Dye labelled oligonucleotides should be stored in aliquots in the dark at -20 °C.Freeze thawing should be avoided.

2. Background level of the probe is very high.

Probes can be degraded separating the fluorophore from quencher, leading toa high background level of the fluorophore. When aliquoting theoligonucleotides sterile tubes and tips must be used to avoid contamination withDNAse enzymes.

Tips: Eurogentec offers you the best fluorescent probes developed from ourreknown high-quality OliGold™ standard.

Background may be due to low efficiency quenching.

Tips: Especially when using long-wave emitting dyes (such as Cy5™), quenchingobtained with Methyl Red or DABCYL is insufficicent. Eurogentec's newElleQuencher™ is the best alternative to solve this problem.

3. SYBR Green™ I reaction has stopped working.

Once SYBR Green™ I has been diluted into T.E. it goes off very quickly and canonly be kept in the fridge for approximately 2 weeks. It should also be kept inthe dark. There are some kits now being sold with SYBR Green™ I diluted intoa solution of DMSO as this appears to keep for longer.

Tips: Eurogentec's Real-Time kits with SYBR Green™ I contain DMSO and willensure you get the best results.

4. No template controls give a positive result.

The master mix may be contaminated with DNA template or PCR product. Cleanworking practises should be used and an Ung step introduced providing dUTPhas been used in the dNTP mix. An alternative is that there is an excess of theprobe and the positive result is an artefact of this. This can be assessed if byusing a no amplification control where the taq is left out. A positive result willthen suggest an artefact and different reagent concentrations can be used.

Tips: To avoid this, use Eurogentec's "Carryover prevention" UNG reagentswhich contain well-balanced dUTP / dTTP solutions.

5. Allelic Discrimination is poor.

Probe design is bad or the mismatch created is particularly stable. Think aboutusing Scorpions™ or Duplex Scorpions™ probes. For Scorpions™ and MolecularBeacons try optimising the monitoring temperature to obtain better discrimination.

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NS Alternatively redesign the probe length and stem sequence. It may be worth trying

to place the primer over the mutation rather than probe if a design is particularlydifficult. A melt curve can help to indicate at which temperature the bestdiscrimination is obtained. This can also be used to observe the dynamics ofhybridisation probes.

Good spectral resolution is also important for allelic discrimination whenmultiplexing probes. Make sure the spectral calibration file is turned on ifnecessary for your machine.

6. I get a low signal/background ratio when usingMolecular Beacon probes. How can you explain that?

• The most likely reason is contamination by either free fluorophores oroligonucleotides that contain the fluorophore but not the quencher. Thefluorophores can be removed by passage through a Sephadex column. In orderto ensure that every molecule contains a quencher, repeat the purification ofoligonucleotides that are protected by a trityl moiety and labeled with DABCYLprior to coupling with the fluorophore.

• The assay medium may contain insufficient salt. There should be at least 1 mMMgCl2 in the solution in order to ensure that stem hybrids form.

• The Molecular Beacon may fold into an alternate conformation that results ina sub-population that is not quenched well. Change the stem sequence (andprobe sequence, if necessary) to eliminate that possibility. Incomplete restorationof fluorescence at low temperatures.

• If the stem of a molecular beacon is too strong, at low temperatures it mayremain closed while the probe is bound to the target. This may happeninadvertently if the probe sequence can participate in the formation of a hairpinthat results in a stem longer and stronger than originally designed. Change thesequence at the edges of the probe and the stem sequence to avoid this problem.

Tips: Always choose Eurogentec’s Molecular Beacon probes and get the bestcustomer support for your design.

7. What's the best alternative to multiplex a Double-Dye PCR assay ?

Multiplexing is crucial when it is necessary to detect different targets in a singleassay. Currently, the most frequently used dye combination for detecting twotargets is FAM and HEX (or JOE but it is expensive) with ROX as the referencedye. Unfortunately, using FAM/HEX or FAM/JOE often leads to less sensitivity.

Tips: Eurogentec proposes the Yakima Yellow™ dye as a replacement for theVIC™ and JOE dyes currently used with most Q&Q PCR systems. The new Yellowdye features improvement in fluorescent signal strength, spectral resolution andproduction cost. The increased signal strength of Yakima Yellow™ dye results ingreater sensitivity and, therefore, lower Ct values at lower ampliconconcentrations.

8. Which dye label should I use for my target andcontrols?

Tips: For optimal results, always choose FAM for your target of interest andEurogentec’s new Yakima Yellow™ dye for your controls.

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KITS AND CONSUMABLES

For 96-well plate systemsWith ROX passive reference

qPCR MasterMix RT-QP2X-03 300 reactions

qPCR Core kit RT-QP73-05 500 reactions

qPCR MasterMix for SYBR Green™ I RT-SN2X-03 300 reactions

qPCR Core kit for SYBR Green™ I RT-SN73-05 500 reactions

qPCR MasterMix PRT RT-QP2X-075+ 300 reactions RT-QP2X-15+ 600 reactionsRT-QP2X-50+ 2000 reactions

Without ROX passive reference

PCR MasterMix No ROX RT-QP2X-03-NR 300 reactions

qPCR Core kit No ROX RT-QP73-05-NR 500 reactions

qPCR MasterMix for SYBR Green™ I No ROX RT-SN2X-03-NR 300 reactions

qPCR Core kit for SYBR Green™ I No ROX RT-SN73-05-NR 500 reactions

For capillary system

Lithos qPCR MasterMix - 2 mM MgCl2 RT-SN73-35LC2 350 reactions

Lithos qPCR MasterMix - 3 mM MgCl2 RT-SN73-35LC3 350 reactions

Lithos qPCR MasterMix - 3 mM MgCl2 RT-SN73-35LC4 350 reactions

Lithos qPCR MasterMix - 5 mM MgCl2 RT-SN73-35LC5 350 reactions

Lithos qPCR Optimisation MasterMix RT-SN73-OKLC 4 x 50 reactions

Lithos qPCR MasterMix Hot Start - 2 mM MgCl2 RT-SNHS-35LC2 350 reactions

Lithos qPCR MasterMix Hot Start - 3 mM MgCl2 RT-SNHS-35LC3 350 reactions

Lithos qPCR MasterMix Hot Start - 4 mM MgCl2 RT-SNHS-35LC4 350 reactions

Lithos qPCR MasterMix Hot Start - 5 mM MgCl2 RT-SNHS-35LC5 350 reactions

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DUCTS For Smart Cycler®

Smart kit RT-QP2X-CE 500 reactions

Smart kit for SYBR Green™ 1 RT-SN2X-CE 500 reactions

25 µl Smart Cycler® tubes RT-PL25-025 5 x 50 tubes

100 µl Smart Cycler® tubes RT-PL00-025 5 x 50 tubes

25 µl Smart Cycler® tubes RT-PL25-100 20 x 50 tubes

100 µl Smart Cycler® tubes RT-PL00-100 20 x 50 tubes

Consumables

Carryover prevention

UNG (Uracyl N-Glycosylase) ME-0610-03 300 units

UNG (Uracyl N-Glycosylase) ME-0610-15 1500 units

Control kits

23S E.coli Genomic Control RT-QP73-23S 250 reactions

ß-actin Genomic Control RT-QP73-BACTIN 250 reactions

GAPDH Genomic Control RT-QP73-GAPDH 250 reactions

Plates for 96-well qPCR systems

qPCR 96-well plate natural RT-PL96-01N 4 X 5 plates + caps

qPCR 96-well plate black RT-PL96-01B 4 X 5 plates + caps

R2T2 REVERSE TRANSCRIPTASE KITS

For instruments requiring passive reference

Probe technology • One-step

Reverse Transcriptase qPCR MasterMix RT-QPRT-032X 300 reactions

Probe technology • Two-step

qPCR Reverse Transcriptase Core kit + qPCR Core kit RT-QPRT-05 500 reactions

SYBR Green™ I • Two-step

qPCR Reverse Transcriptase Core kit+ qPCR Core kit for SYBR Green™ I RT-SNRT-05 500 reactions

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DUCTSFor Smart Cycler®

Probe technology • One-step

Reverse Transcriptase qPCR MasterMix NO ROX RT-QPRT-032XNR 300 reactions

Probe technology • Two-step

qPCR Reverse Transcriptase Core kit + qPCR Core kit NO ROX RT-QPRT-05NR 500 reactions

SYBR Green™ I • Two-step

qPCR Reverse Transcriptase Core kit+ qPCR Core kit for SYBR Green™ I NO ROX RT-SNRT-05NR 500 reactions

FLUORESCENT PROBES

Molecular Beacon probes5’ (FAM/HEX/TET) + 3’ DABCYL OL-0371-0802 0.2 µmol

5’ (FAM/HEX/TET) + 3’ DABCYL OL-0371-0810 1 µmol

5’ Cy Dyes™ + 3’ DABCYL OL-0371-0803 0.2 µmol

5’ (Rhodamine/JOE/ROX)+ 3’ DABCYL OL-0371-0804 0.2 µmol

5’ TAMRA + 3’ DABCYL OL-0371-0804 0.2 µmol

5’ Cascade Blue + 3’ DABCYL OL-0371-0804 0.2 µmol

5’ Bodipy + 3’ DABCYL OL-0371-0804 0.2 µmol

5’ Alexa + 3’ DABCYL OL-0371-0809 0.2 µmol

2’ O-Me RNA Molecular Beacon (HEX/TET) OL-0371-0820 0.2 µmol

New quenchers (ElleQuencherTM : EQ – Eclipse™ Dark Quencher : DQ)

5’ (HEX/TET) + 3’ DQ OL-0371-0811 0.2 µmol

5’ Cy Dyes™ + 3’ EQ OL-0371-0812 0.2 µmol

5’ (Rhodamine/JOE/ROX) + 3’ EQ OL-0371-0813 0.2 µmol

5’ Alexa + 3’ EQ OL-0371-0814 0.2 µmol

5’ TAMRA+ 3’ EQ OL-0371-0816 0.2 µmol

Wavelength-shifting Molecular Beacon probes

WSB Molecular Beacon (h:fluorescein, e: TAMRA, q: DABSYL)§ OL-0372-WSBO1 0.2 µmol

WSB Molecular Beacon (h:fluorescein, e: TAMRA, q: ElleQuencherTM)§ OL-0372-WSBO3 0.2 µmol

(§) h: harvester fluorophore; e: emitter fluorophore; q: quencher. Other combinations of fluorophores areavaible upon request.

Eurogentec belongs to the licensed providers of Molecular Beacons.

Prices include licence fee, oligonucleotide synthesis and purification.

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DUCTS Double-Dye Oligonucleotide probes

5’ FAM + 3’ TAMRA OL-0361-0802 0.2 µmolOL-0361-0810 1 µmol

5’ (HEX/TET) + 3’ TAMRA OL-0361-HEXTAM02 0.2 µmol

5’ FAM + 3’ DABCYL OL-0361-TADA 0.2 µmol

5’ TAMRA + 3’ DABCYL OL-0361-TADA 0.2 µmol

5’ FAM + 3’ DQ OL-0361-FAMDQ02 0.2 µmol

5’ Yakima Yellow™ + 3’ DQ OL-0361-YADQ02 0.2 µmol

5’ Cy Dyes™ + 3’ EQ OL-0361-CYEL02 0.2 µmol

5’ TAMRA+ 3’ EQ OL-0361-TAMEL02 0.2 µmol

Prices include oligonucleotide synthesis and purification

Scorpions™ probesScorpions™ probe (include oligonucleotide synthesis) OL-0361-S802

FRET probes for capillary system

3'FAM OL-0390-LCFAM02 0.2 µmol

5'ROX OL-0390-LCROX02 0.2 µmol

5’ Cy5™ OL-0390-LCCY02 0.2 µmol

Prices include oligonucleotide synthesis and purification

FLUORESCENT DYES AND QUENCHERSThese oligonucleotides are always purified. Minimum delivered quantity : 100 µg; max 30 bases/oligonucleotide.

5'-HEX OL-0351-02 0.2 µmol 150 µgOL-0351-10 1.0 µmol 400 µg

5'-TET OL-0352-02 0.2 µmol 150 µgOL-0352-10 1.0 µmol 400 µg

5'-FAM OL-0353-02 0.2 µmol 150 µgOL-0353-10 1.0 µmol 400 µg

5'-ROX OL-0354-02 0.2 µmol 100 µgOL-0354-10 1.0 µmol 300 µg

5'-TAMRA OL-0355-02 0.2 µmol 100 µgOL-0355-10 1.0 µmol 300 µg

5'-JOE OL-0356-02 0.2 µmol 100 µgOL-0356-10 1.0 µmol 300 µg

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DUCTS3'-TAMRA OL-0357-02 0.2 µmol 100 µg

OL-0357-10 1.0 µmol 300 µg

TAMRA-dT OL-0357-DT02 0.2 µmol

TAMRA NH ESTER OL-0357-NHS02 0.2 µmol

5'-Cy5™ OL-0CY5-02 0.2 µmol 150 µgOL-0CY5-10 1.0 µmol 400 µg

5'-Cy5.5™ OL-0CY5-502 0.2 µmol 150 µg

5'-Cy3™ OL-0CY3-02 0.2 µmol 150 µgOL-0CY3-10 1.0 µmol 400 µg

5’Yakima Yellow™ OL-0359-02 0.2 µmol

3'DABCYL OL-0273-0302 0.2 µmol

3'DABCYL OL-0273-0310 1.0 µmol

3’ElleQuencher™ OL-0273-EL02 0.2 µmol

3’Eclipse™ Dark Quencher OL-0273-DQ02 0.2 µmol

SMART CYCLER® EQUIPMENT

Smart Cycler® integrated DNA/RNA amplification and detection instrument systems

16-site block • Computer and software • USB cable • Accessory pack including: 1 mini-centrifuge + 4 reaction tube raks + 1 cooling block + software + users manual EQ-0001-SC

Additional unit including processing block EQ-0002-SC

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JARG

ON Jargon

Amplicon: Fragment of DNA produced from a pair of primers during PCR. Thefragment is of a specific sequence and length.

Amplifluor™: Non-specific PCR detection method that employs the ability of taqto open out a hairpin loop structure to cause an increase in fluorescence as thefluorophore and quencher are separated.

Background fluorescence: Refers to the fluorescence inherent in an assay orexperiment due to the inefficient quenching of the reporter dye or spurious sidereactions.

cDNA: Complementary DNA produced by reverse transcription of RNA

CFTR: Cystie fibrosis transmembrane conductance regulator

Ct: First cycle at which the fluorescent signal obtained during Q&Q PCR issignificantly higher than the background signal.

Dark Quencher: Molecule that quenches a fluorophore and emits the energy asheat rather than fluorescing itself.

∆G: Change in free energy. Negative values indicate that energy is emitted bya reaction and positive values indicate that energy is needed for a reaction tooccur.

Double-Dye Oligonucleotide probe: Specific detection probe that binds to anamplicon during PCR. It uses the 5’-3’ exonuclease activity of the taq to degradethe probe. This releases the fluorophore and quencher from either end of theprobe so that the fluorophore is no longer quenched through FRET with thequencher.

Emission maxima: Refers to the wavelength in greatest abundance among thewavelengths emitted from a fluorescent molecule. This wavelength is shown asthe highest point on an emission peak.

Emission wavelength: Refers to the wavelength of light produced by an excitedmolecule as it returns to an non-excited state.

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JARG

ONEndogenous Control: a cellular RNA used as an internal control template. The

control is used to normalise experimental data and should be unaffected byexperimental regimes. ß-actin and GAPDH are 2 commonly used controls.

Exogenous Control: External control DNA that is spiked into the experimentalsample. This is used to control for PCR inhibitors and pipetting errors. This typeof control is also often used as a standard for absolute quantification.

Fluorophore: A class of molecules called reporter dyes that emit or fluorescelight of a particular wavelength or a result of being irradiated with light ofanother particular wavelength that excites the molecule.

Free-energy difference: The difference in the relative energy associated withthe hybridization of two different energetic systems. In this case, the two systemsare the hybridization of two different nucleic acid complements.

FRET: Fluorescence Resonance Energy Transfer. Energy from one fluorophore istransmitted to another fluorophore with an excitation spectra that overlaps withthe emission spectra of the first fluorophore. The second fluorophore emits thefluorescence at a different wavelength.

Melt Curve: Curve generated by slowly increasing the temperature of a PCRreaction following amplification, whilst continuously monitoring fluorescence.Changes in fluorescence can be observed due to melting of probe-targetduplexes or the melting of the double stranded amplicon and drop in SYBR Green™ I fluorescence. Melting peaks can be obtained by when plottingthe first negative derivative of fluorescence vs. temperature. This is often used toobserve the Tm of probes and for mutation detection. An alternative to using thePCR reaction in order to produce a melt curve is to make an artificial target forthe probe.

Molecular Beacon: Probe that is held in a hairpin loop structure. The loop holdsa fluorophore and quencher in close proximity. Binding to an amplicon of thecorrect sequence causes the hairpin to open and fluorescence increases.

mRNA: Messenger RNA. RNA that is translated to proteins in the cell.

Non-fluorescent: A term meaning that when irradiated with light, thesemolecules do not become excited and emit light themselves.

Quencher: Molecule that is able to absorb the energy emitted by a fluorophore,through FRET (see below). It emits the energy at a different wavelength, reducingfluorescence of the fluorophore. Alternatively the quencher may act throughcollisional quenching, where the fluorophore and quencher are on close contact.

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JARG

ON ∆Rn: Normalised reporter signal. Calculated by dividing the reporter signal

(minus baseline) by the passive reference signal.

rRNA: Ribosomal RNA, found in ribosomes in the cell.

Scorpions™: Intramolecular sequence-specific probe. The probe is held in ahairpin loop that holds the fluorophore and quencher together. This is linked toa PCR primer by a PCR stopper. On extension of the primer the probe opens outand binds to the extension product in a unimolecular manner.

SYBR Green™ I: non-specific double stranded DNA intercalating dye thatfluoresces when bound to the DNA. It is often used as the Q&Q PCR equivalentto ethidium bromide, detecting all double stranded DNA products in a PCRreaction, including primer dimers.

Tm: Melting temperature of a probe or primer, i.e. the temperature at which 50 % of the oligonucleotide is bound.

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REF

EREN

CESReferences

Bustin S. (2000). Absolute quantification of mRNA using Real-Time reversetranscription polymerase chain reaction assays. Journal of Molecular EndocrinologyVol. 25, p169-193.

Caplin B.E., Rasmussen R.P., Bernard P.S. and Wittwer C.T. (1999). LightCycler™Hybridisation Probes - The most direct way to monitor PCR amplification and mutationdetection. Biochemica Vol. 1, p5-8.

Freeman W. M., Walker S. J., Vrana K. E. (1999) Quantitative RT-PCR: pitfalls andpotential. Biotechniques Vol. 112-22, p124-125.

Halford W. P. (1999) The essential prerequisites for quantitative RT-PCR. NatBiotechnol, Vol. 17 (19), p835.

Heid C. A., Stevens J., Livak K. J., Williams P. M. (1996) Real time quantitative PCR.Genome Res. Vol. 6 (10), p986-94.

Holland P., Abramson R.D., Watson R. and Gelfand D.H. (1991). Detection ofspecific polymerase chain reaction product by utilizing the 5’-3’ exonuclease activityof thermus aquaticus. Proc. Natl, Acad, Sci. USA.Vol. 88, p7276-7280.

Ju J., Ruan C., Fuller C.W., Glazer A.A. and Mathies R.A. (1995). Fluorescenceenergy transfer dye-labeled primers for DNA sequencing and analysis. Proc. Natl.Acad. Sci. USA. Vol. 92, p4347-4351.

Kutyavin I. V., Afonina I. A., Mills A., Gorn V. V., Lukhtanov E. A., Belousov E. S.,Singer M. J., Walburger D. K., Lokhov S. G., Gall A. A., Dempcy R., Reed M. W.,Meyer R. B., Hedgpeth J. (2000) 3'-minor groove binder-DNA probes increasesequence specificity at PCR extension temperatures. Nucleic Acids Res. Vol. 28;p655-61.

Landt O. and Nitsche A. LightCycler™ Technical Note. Selection of HybridisationProbe Sequences for Use with the LightCycler™. www.TIB-MOLBIOL.de

Molenaar C, Marras SA, Slats JC, Truffert JC, Lemaitre M, Raap AK, Dirks RW,Tanke HJ. Linear 2' O-Methyl RNA probes for the visualization of RNA in living cells.Nucleic Acids Res. 2001 Sep 1;29(17):E89-9.

Mullis K. and Faloona F. (1987) in Methods in Enzymology, Vol. 155, p335 (Ed. R.Wu).

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Nasarabadi S., Milanovich F., Richards J. and Belgrader, P. (1999). Simultaneousdetection of TaqMan probes containing FAM and TAMRA reporter fluorophores.BioTechniques Vol. 27(6), p1116-1117.

Solinas A., Brown L. J., McKeen C., Mellor J. M., Nicol J., Thelwell N., Brown T.(2001) Duplex Scorpion primers in SNP analysis and FRET applications. NucleicAcids Res. Vol. 29, E96 .

Suzuki T., Higgins P. J., Crawford D. R. (2000) Control selection for RNAquantitation. Biotechniques. Vol. 29, p332-7.

Svanvik, N., Ståhlberg A., Sehlstedt U., Sjöback R. and Kubista M. (2000).Detection of PCR Products in Real Time Using Light-up Probes. Analytical BiochemistryVol. 287, p179-182.

Szuhai K, Ouweland J, Dirks R, Lemaitre M, Truffert J, Janssen G, Tanke H, HolmeE, Maassen J, Raap A. Simultaneous A8344G heteroplasmy and mitochondrial DNAcopy number quantification in myoclonus epilepsy and ragged-red fibers (MERRF)syndrome by a multiplex molecular beacon based real-time fluorescence PCR.Nucleic Acids Res. 2001 Feb 1;29(3):E13.

Szuhai K, Sandhaus E, Kolkman-Uljee SM, Lemaitre M, Truffert JC, Dirks RW,Tanke HJ, Fleuren GJ, Schuuring E, Raap AK. A novel strategy for humanpapillomavirus detection and genotyping with SybrGreen and molecular beaconpolymerase chain reaction. Am J Pathol. 2001 Nov;159(5):1651-60.

Thelwell N., Millington S., Solinas A., Booth J. and Brown T. (2000). Mode ofaction and application of Scorpion primers to mutation detection. Nucleic AcidsResearch Vol. 28(19), p3752-3761.

Tyagi S. and Kramer F.R. (1996). Molecular Beacons: Probes that fluoresce uponhybridization. Nature Biotechnology Vol. 14, p303-308.

Tyagi, S., Marras, S.A.E. and Kramer, F.R. (2000). Wavelength-shifting MolecularBeacons. Nature Biotechnology Vol. 18, p1191-1196.

Whitcombe D., Theaker J., Guy S.P., Brown T. and Little S. (1999). Detection ofPCR products using self-probi nM amplicons and fluorescence. Nature BiotechnologyVol. 17, p804-807.

Wolffs P., Knutsson R., Sjoback R., Radstrom P. (2001) PNA-Based light-up probesfor real-time detection of sequence-specific PCR products. Biotechniques. Vol. 766,769-71.

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NOTICES

The Polymerase Chain Reaction (PCR) process is covered by patents owned by Hoffmann-La Roche, Inc. Use of the PCRprocess requires a license.

A license under U.S. Patents 4,683,202, 4,683,195 and 4,965,188 or their foreign counterparts, owned by RocheMolecular Systems, Inc. and F. Hoffmann-La Roche Ltd (“Roche”), has an up-front fee component and a running-royaltycomponent. The purchase price of this product includes limited, nontransferable rights under the running-royalty componentto use only this amount of the product to practice the Polymerase Chain Reaction ("PCR") and related processes describedin said patents solely for the research and development activities of the purchaser when this product is used in conjunctionwith a thermal cycler whose use is covered by the up-front fee component. Rights to the up-front fee component must beobtained by the end user in order to have a complete license. These rights under the up-front fee component may bepurchased from Applied Biosystems or obtained by purchasing an Authorized Thermal Cycler. No right to perform or offercommercial services of any kind using PCR, including without limitation reporting the results of purchaser's activities for afee or other commercial consideration, is hereby granted by implication or estoppel. Further information on purchasinglicenses to practice the PCR Process may be obtained by contracting the Director of Licensing at Applied Biosystems, 850Lincoln Centre Drive, Foster City, California 94404 or the Licensing Department at Roche Molecular Systems, Inc., 1145Atlantic Avenue, Alameda, California 94501.

The use of certain fluorogenic probes in 5' nuclease assays is covered by US patents 5,210,015 and 5,487,972, ownedby Roche Molecular Systems, inc, and by US patent 5,538,848, owned by Applied Biosystems. Purchase of this productdoes not provide a license to use this patented technology. A license must be obtained by contracting the Director ofLicensing Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404 or the Licensing Department at RocheMolecular Systems Inc., 1145 Atlantic Avenue, Almeda, CA 94501.

Certain products of Eurogentec S.A. are accompanied by a limited license to use them in the PCR process for life scienceresearch with a thermal cycler whose use in the automated performance of the PCR process is covered by the up-front licensefee, either by payment to Applied Biosystems or as purchased, i.e. and authorized thermal cycler.

No rights under US Patents 5,210,015 and 5,847,972 are hereby conveyed.

Use of UNG employs patent rights licensed to Eurogentec s.a. from Invitrogen Corporation.

Cyclic-substituted unsymmertical cyanine dyes are covered by US patent 5,436,134 and 5,658,751 and licensed toEurogentec by Molecular Probes in the direct research field.

The use of ScorpionsTM Probes for research purposes is covered by a license to Eurogentec S.A. from DxS Ltd. No rights toperform or offer diagnostic, commercial testing or other commercial services for money or money's worth are granted bythe supply of this product. Should you wish to use this product for any other purpose not covered by this license, pleasecontact DxS Ltd at Manchester Incubator Building, 48 Grafton Street, Manchester, M13 9XX.

The use of Molecular Beacon probes employs patent rights licensed to Eurogentec S.A. from The Public Health ResearchInstitute of the City of New York and is limited to research purposes only. The use of ElleQuencherTM is covered by patentapplication owned by Eurogentec s.a.

Elle QuencherTM, OliGoldTM, GoldStarTM and HotGoldStarTM are trademarks of Eurogentec s.a.SYBR GreenTM, SYBR GoldTM and Texas Red™ are trademarks of Molecular Probes Inc.Cy™ and Cy Dye™ are trademarks of Amersham Life SciencesEclipseTM and Yakima YellowTM are trademarks of Synthetic GeneticsABI PrismTM, VICTM and NEDTM are trademarks of Applied BiosystemsRed 640 and Red 705 are licensed from Hoffman La Roche Ltd.AmplifluorTM and UniprimerTM are trademarks of IntergenDNA Engine OpticonTM is a trademark of MJ ResearchRiboGreen® is registered by Molecular Probes Inc.OligoTM is a trademark of Molecular Biology Insights Inc.Clontech TaqStartTM is a trademark of BD BiosciencesICyclerTM is a trademark of Bio-Rad LaboratoriesSmart Cycler ® is registered by CepheidScorpionsTM is a trademark of DxS Ltd.MX-4000TM is a trademark of StratageneHy-BeaconsTM is a trademark of LGCI-CORETM is a trademark of Cepheid

NO

TICES

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SUBSIDIARIES:

BELGIUMEUROGENTEC s.a.Tel.: 04 366 61 00Fax: 04 264 07 88

FRANCEEUROGENTEC France s.a.Tél.: 02 41 73 33 73Fax: 02 41 73 10 26

GERMANYEUROGENTEC Deutschland GmbHTel.: 0221 258 94 55Fax: 0221 258 94 54

THE NETHERLANDSEUROGENTEC Nederland b.v.Tel.: 043 363 40 37Fax: 043 363 77 65

SWITZERLANDEUROGENTEC s.a. succursale de GenèveFrançais:Tél.: + 32 4 366 61 00Fax: + 32 4 264 07 88Deutsch:Tel.: + 49 221 258 94 55Fax: + 49 221 258 94 54

UNITED KINGDOMOSWEL RESEARCH PRODUCTS LtdTel.: 023 80 59 29 84/85Fax: 023 80 59 29 82/83

JAPANNIPPON EGT k.k.Tel.: 076 411 02 77Fax: 076 452 03 99

U.S.A.See www.eurogentec.com

Headquarters:Parc scientifique du Sart Tilman

4102 Seraing - BELGIUMTel.: +32 4 366 61 00Fax: +32 4 365 16 04

[email protected] - www.eurogentec.com

DISTRIBUTORS:

NORWAY/SWEDENFINLAND/DENMARKMedProbe ASTel.: + 47 23 32 73 80Fax: + 47 23 32 73 90

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ITALYBiosense srlTel.: 02 61 25 911Fax: 02 61 25 933

SPAINCultek s.l.Tel.: 091 729 03 33Fax: 091 358 17 61

PORTUGALMerck EurolabTel.: 021 361 36 20Fax: 021 362 56 15

JAPANNippon Gene Co. LtdTel.: 076 451 65 48Fax: 076 451 65 47

ISRAELDexmorTel.: 03 649 85 03Fax: 03 649 80 41

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