Lal:§9_nsumer
`
`Up to Speed on PCR
`
`
`
`Real—tirne systems enable
`fast, quantitative analyses
`Bv Danonnu A. Fn-zcsnnua
`
`CR—a technique so common in
`Ptoday’s laboratoriesthatit is easy to
`forget
`its
`revolutionary impact-
`enables the specific amplification and
`detection of as little as a single copy of a
`particular nucleotide sequence. However,
`PCR has the potential to be used not just
`for the detection of specific sequences,
`but also for their quantification, because
`of the quantitative relationship between
`the amount of starting target sequence and
`the amount of PCR product at any given
`cycle that falls within the reaction’s expo-
`nential range.
`The theory is straightforward, but a
`number of technical caveats are associat—
`ed with the use of conventional end-point
`methodologies for quantitative PCR.”
`In these techniques, PCR results are
`monitored after a given number of
`cycles, by which point factors such as
`limiting reagent concentrations and side
`reactions may have played a significant
`role in affecting final product concentra»
`tion. Quantitative competitive PCR was
`developed in response to some of these
`difficulties. In this approach, the starting
`amount of target is calculated based on
`
` the ratio of target to competitor after
`amplification. However, quantita—
`tive competitive PCR is
`cumbersome, and it can be
`associated with a number
`of drawbacks including a
`limited dynamic range and
`the need to screen multiple
`dilutions.”
`Some of the limitations
`of end—point PCR have
`been assuaged in real-time
`PCR systems, a number of
`which are now on the market.
`These systems offer many
`general technical advantages,
`including reduced probabili-
`ties of variability and con-
`tamination. as well as
`online monitoring and
`the lack of need for
`postreaction analy-
`ses. Further, some of
`these systems were
`developed with con-
`temporary
`applications
`such as quantitative PCR,
`multiplexing, and high—throughput
`(HT) analysis in mind? In real-time
`quantitative PCR techniques, signals
`generally fluorescent) are monitored as
`they are generated and are tracked after
`they rise above background but before
`the reaction reaches a plateau.
`Initial
`template levels can be calculated by ana~
`lyzing the shape of the curve or by deter-
`mining when the signal rises above some
`threshold value.1»Z Several commercially
`
`
`
`eral general types of fluorescent probes1
`for detection.
`Several different basic types of fluoe
`rescent probes are used for real—time PCR
`applications. Some assays employ general
`probes that bind preferentially to double-
`stranded DNA (as opposed to the single-
`stranded template). Others use target
`sequence-specific reagents such as
`exonuclease probes. hybridiza-
`tion probes, or molecular bea-
`cons (hairpin probes). Although
`more
`expensive,
`sequence~
`specific probes add specificity to
`the assay. and enable multiplex-
`ing applications.
`Fmsr ON Lms
`Applied Biosystems of
`Foster City, Calif., was
`the first to commercial—
`ize real-time PCR. This
`company offers
`the
`ABI PRISM® 7700
`Sequence Detection
`System, which inte-
`grates a closed—tube
`PCR»based
`assay
`with instrumentation and
`software that enable real-time quantita-
`tive PCR. This system is optimized for
`use of a fluorogenic 5’ exonuclease assay
`that employs the Thermus aquaticus
`(Taq) polymerase. Applied Biosystems
`
`avail—
`able real—
`time
`PCR
`systems
`are
`overviewed in this article and/or summa-
`rized in the accompanying table. Each of
`these systems employs either one of sev-
`
`
`
`requisite
`the
`commercialized
`has
`reagents in its well-known TaqMan®
`product
`line. The fluorogenic probe is
`complementary to the target sequence,
`and initially contains both reporter and
`quencher moieties. When the probe is
`not bound to template DNA, its reporter
`and quencher dyes are in close proximi—
`ty, and the reporter’s fluorescent emis-
`sion is quenched.
`The probe is designed to anneal
`specifically between the forward and
`reverse primer
`sites of
`the
`target
`sequence. If a template bearing the target
`sequence is present. the probe anneals to
`it. During PCR, the nuclease activity of
`Taq polymerase cleaves the reporter dye
`from the probe. The reporter dye, now
`separated from the quencher, emits a flu-
`orescent signal. Thus, fluorescent signal
`is emitted only after the probe binds
`template DNA and is cleaved during the
`course of PCR. The fluorescent signal is
`monitored at every cycle as additional
`reponer dye molecules accumulate. After
`the signal rises above background,
`its
`rate of increase is tracked during a num-
`ber of linear cycles before the reaction
`reaches a plateau. These data are then
`used to calculate initial template levels.
`The ABI PRISM 7700 was designed
`with HT applications in mind. Reactions
`are perfomied in 96»wel1 microplates,
`and it takes merely 15 seconds for this
`
`The Scientist 3 1
`November 2 7, 2000
`
`THERMO FISHER EX. 1034
`THERMO FISHER EX. 1034
`
`

`
`LabConsumer
`
`Real-time PCR Systems
`
`ABI PRISM 7700 Sequence Detection system
`
`Microplate (96)
`
`37
`
`24
`
`27
`
`320 lbs.
`
`Yes
`
`'
`
`Arman Biosvsrms
`(800) 345-5224, www.app|iedbiosystems.c0m
`BIo-RAD
`(800) 4-BIDRAD, wwwhio-radmrn
`
`it cler iQ Real-Time PCR Detection System
`(thermal Cycler plus optimal optical module)
`
`Microplate (96), Tubes (96)
`
`1-
`
`-.6
`x"
`
`,_
`
`»,a_
`
`.
`
`'12
`
`_
`
`i.
`
`.4.
`
`.
`
`_,
`
`_
`
`-.
`
`.
`
`' distributed by Fisher Scientific, (800) 766-7000. www.fishersci.com
`1 dimensions given in inches
`
`system to collect one round of fluores-
`cent emission data from each well. The
`reaction tube's transparent
`lid allows
`laser light, which is carried on an array
`of optical fibers, to be distributed to each
`well. The laser light excites the reporter
`dye molecules to fluoresce, and the
`resultant signals are carried by the optic
`fibers to a charge-coupled device (CCD)
`camera
`for detection. Applications
`include allelic discrimination. melting
`curves, and quantification of single or
`multiplexed targets.
`Gomc LIGHT
`Roche
`LightCycler"“
`from
`The
`Molecular Biochemicals of Indianapolis
`harnesses light technology licensed from
`Idaho Technology Inc. of Idaho Falls,
`and then adds some additional bells and
`whistles, such as a built—in microvolume
`fluorimetric
`detection
`system that
`enables real-time quantitative PCR. This
`system employs thin—waJled glass capil-
`lary tubes, and typical PCR experiments
`can be performed in less than 30 min-
`utes. Currently, this system supports two
`fluorescence-based methods
`for
`the
`detection of amplification products: the
`“genera_l" DNA stain SYBR Green I (a
`product of Molecular Probes of Eugene,
`Ore.), or sequence-specific hybridization
`probe pairs.
`SYBR Green I exhibits very little flu-
`orescence when free in solution; emis-
`sion is greatly enhanced when it binds to
`the minor groove of the DNA double
`helix. Prior to amplification, the reaction
`mixture contains the denatured DNA, the
`primers, and the dye. The low—level
`background fluorescence signal generat-
`ed by the unbound dye molecules is sub-
`tracted during computer analysis. After
`annealing of the primers, a few dye mol-
`ecules can bind to the double strand.
`During elongation, more and more dye
`molecules bind to the newly synthesized
`DNA, resulting in dramatically increased
`light emission. If the reaction is moni-
`tored continuously, this increase in fluo-
`rescence can be viewed in real
`time.
`After denaturation of the DNA during
`
`32 _The Scientist
`November 27, 2000
`
`The sequences of the two oligonu-
`cleotides are selected so that
`they
`hybridize to the amplified DNA frag-
`ment
`in a head-to—tail arrangement.
`When the oligonucleotides hybridize in
`this orienmtion, the two fluorescent dyes
`are positioned in close proximity to each
`other. The first dye (fluorescein) is excit-
`ed by the LightCycler’s light emitting
`diode (LED) filtered light source, and
`emits green fluorescent light at a slightly
`longer wavelength. When the two dyes
`are in close proximity, the emitted ener-
`gy excites the dye attached to the second
`hybridization probe, which subsequently
`emits red fluorescent
`light at an even
`longer wavelength. This energy transfer,
`referred to as
`fluorescence resonance
`energy transfer (FRET), occurs efficient-
`ly only when the dyes are in close
`proximity (a distance between
`1-5
`nucleotides). Thus, in this type of assay,
`fluorescent
`intensity measurements are
`made after
`the annealing steps. The
`increasing amount of emitted fluores-
`cence is proportional
`to the increasing
`amount of DNA generated during the lin-
`ear phase of the ongoing PCR process.
`Con: MODULE
`The “heart” of the Smart Cycler-® System
`from Cepheid of Sunnyvale, Calif, is the
`I-CORE” (Intelligent Cooling/Heating
`Optical Reaction) module. According to
`company literature, the I-CORE module
`incorporates state-of—the—an microfluidic
`and microelectronic design. Each Smart
`Cycler processing block contains
`16
`independently programmable I-CORE
`modules, each of which performs four-
`color, real—time fluorometric detection. A
`wide variety of different multiplex or
`simplex fluorescent tags can used in con-
`junction with this system,
`including
`FAM, TET, TAM, ROX, SYBR Green,
`Cy3, Alexa, and Texas Red.
`Samples are amplified and measured in
`proprietary, sealable reaction tubes that are
`designed to optimize rapid thermal transfer
`and optical sensitivity. The Smart Cycler
`software enables single or multiple opera-
`tors to define and simulmneously carry out
`
`The Lightcycler System from Roche
`
`the next heating cycle, the dye molecules
`are released and the fluorescence signal
`falls. A fluorescence measurement is per-
`formed at the end of the elongation step
`of every PCR cycle to monitor
`the
`increasing amount of amplified DNA.
`The hybridization probe
`format
`employs
`two
`specially
`designed,
`sequence-specific oligonucleotides lab-
`
`eled with fluorescent dyes. One oligonu-
`cleotide probe carries a fluorescein label
`at its 3' end; the other probe carries a dif-
`ferent label (LC Red 640 or LC Red 705)
`at its 5‘ end. The chemical nature of the
`hybridization probes prevents
`their
`extension: one probe contains fluores-
`cein at the 3' end, whereas the 5‘—labeled
`probe contains a 3' phosphate moiety.
`
`THERMO FISHER EX. 1034
`THERMO FISHER EX. 1034
`
`

`
`LabConsumer
`
`from companies ofiering fairly comprehen-
`sive quantitative PCR systems that include
`notjust the thermal cycler and optical equip-
`ment, but the requisite computer hardware
`and software as well. The accompanying
`table contains summary information for
`these systems, as well as for additional real-
`
`time PCR instruments that employ user-
`supplied computer hardware. E3
`Deborah Fitzgerald
`(df@ sciwriterrcam) is afreelance
`science writer in Birmingham, Ala.
`References
`
`1.
`
`PE Biosystems. “DNAIRNA Real-Time Quant-
`itative PCR," www.appliedbiosystems.com/
`molecularbiology/aboutlpcr/sds/5700_sds/pdf/
`duarnapdf, 1999.
`E. Zubritsky, "Pinning down PCR: Vlftdespread
`interest i.n gene quantitation and high-throughput
`assays are putting quantitative PCR back in the spot-
`light" Analytical Chemistry, 71 : l9lA—5A, 1999.
`
`
`
`
`
`Software Specifically Designed for
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`
`The Scientist
`November 2 7, 2000
`
`THERMO FISHER EX. 1034
`THERMO FISHER EX. 1034
`
`multiple separate experiments, each with a
`unique set of cycling protocols. In addition,
`thermal and optical data from each and all
`sites can be monitored in real time, and
`graphs of temperature, growth curves, and
`melt curves can be charted as the data are
`collected. The Smart Cycler Starter System
`includes a processing block, Windows-
`compatible computer and monitor, soft-
`ware, mini—centrifuge, tube racks, and a
`cooling block specifically designed to
`accommodate Smart Cycler reaction
`tubes (25 or 100 pl).
`
`Comma Soon
`Stratagene of La Jolla, Calif., plans to
`launch
`the Mx4000” Multiplex
`Quantitative PCR System at the end of
`this year. The Mx4000 combines the
`capabilities of a microplate fluores-
`cence reader with a PCR thermal cycler
`into a single real-time detection system
`and allows detection of multiple fluo-
`rescent PCR chemistries. It includes an
`integrated, proprietary themtal system
`and a mu1tip1e—fluorophore detection
`system. The Mx4000 Multiplex
`Quantitative PCR System has extended
`excitation (350 to 750 nm) and detec-
`tion (35O to 830 nm) ranges, allowing
`researchers to choose fluorophores
`with little or no spectral overlap. Each
`of the four scanning fiber-optic heads
`independently excites and detects dyes,
`reading up to four dyes in a single tube.
`Optimized interference filters are used
`to block out unwanted crosstalk from
`spectrally adjacent tluorophores.
`Researchers can choose from FAM,
`HEX, TAMRA. Cy5, Cy3, Texas
`Red/ROX, and TET filter sets, and cus-
`tom sets for other tluorophores also are
`available. Reactions are performed in
`96-well microtiter plates, but
`res-
`earchers can opt to analyze reactions in
`a subset of the wells rather than always
`reading the entire plate. The system
`includes PCR application software,
`including a feature that enables real-
`timc amplification plots. Thus, the user
`can monitor progress of an experiment
`at any time during thermal cycling,
`rather than waiting until the end of the
`run. Data can be viewed in a variety of
`fomis,
`including amplification plots,
`scatter plots, sample value screens, flu-
`orescence intensity screens, melting
`curves, annealing ranges, and text
`reports.
`The
`system
`employs
`Stratagene’s new solid-state heating
`and cooling technology. According to
`the company’s promotional literature,
`the low thermal mass of the sample
`temperature control block promotes
`rapid changes in temperature, resulting
`in speedy thermal
`ramp rates and
`greater temperature uniformity.
`The
`instruments mentioned here
`enable “closed tube" PCR analysis in real
`time. Results are available during and
`after PCR, with no additional purification
`or analyses. This reduces the likelihood of
`introducing variability or contaminants
`and allows researchers to say goodbye to
`postamplification analytical gels. This
`article focused primarily on products
`
`

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`THERMO FISHER EX. 1034