Real—Time PCR Takes
`Center Stage
`
`Falling instrument prices and a good track record are
`breathing new life into the real-time PCR market.
`Laura DeFrancesco
`
`ntil recently, the real-time poly-
`merase chain reaction (PCR) has
`been on the fringes of molecular biology
`research. There is no question that the
`ability to quantitate nucleic acids using
`this twist on conventional PCR is supe-
`rior to most any other technique, such
`as Northern blots, dot blots, or RNAse
`protection assays. Unfortunately, though,
`the cost of the instruments and the de-
`
`signer probes that some researchers use
`kept this technique out of the hands of
`the average molecular biologist.
`In the last few years, all this has
`changed. According to Dave Ginzinger
`of the University of California—San Fran-
`cisco’s Genome Analysis Core Facility,
`“In the interim of only a couple of years,
`there was a 10-fold increase in the num-
`
`ber of papers—a coming of age of the
`technique.”
`One reason for this change is that
`the standard for affordable instruments
`
`has been raised by various high-priced
`technologies, like automated DNA se-
`quencing or high—density microarrays,
`both of which are commonplace in core
`facilities and in many individual investi-
`gator labs. Real-time PCR machines,
`while still somewhat expensive, are
`priced well below DNA sequencers.
`Furthermore, the entry of several new
`instrument makers into the field, as well
`as new probe designs and chemistries,
`have increased competition and driven
`costs down. Table 1 lists some instru-
`
`ments that are commercially available
`now; this list is not meant to be com-
`prehensive but to indicate the types of
`options available to potential buyers.
`
`Quantitating nucleic acids was the ini-
`tial application and may still be what re-
`searchers most often use it for, but that’s
`not the whole story. According to Scot-
`
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`175A
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`product review
`
`Table 1. Selected real-time PCR instruments.
`
`Product
`
`Company
`
`ABI PRISM 7900HT
`
`Applied Biosystems
`850 Lincoln Centre Dr.
`Foster City, CA 94404
`800-327-3002
`
`ABI PRISM 7000
`
`Applied Biosystems
`850 Lincoln Centre Dr.
`Foster City, CA 94404
`800-327-3002
`
`icycler in
`
`Bio-Rad Laboratories
`2000 Alfred Nobel Dr.
`Hercules, CA 94547
`800-4-BIORAD
`
`URL
`
`www.app|iedbiosystems.com
`
`wvvw.app|iedbiosystems.com
`
`www.bio-rad.com/iCyc|er
`
`List price (U.S.D.)
`
`$90,000; $130,000 with optional automation
`accessory
`
`$47,250
`
`$49,500
`
`Excitation
`
`Detection
`
`Extended-life argon-ion laser and dual-axis Tungsten/halogen lamp
`synchronous scanning head
`
`Tungsten/halogen lamp with five-position fil-
`ter wheel
`
`Spectrograph and cooled CCD; detection of Four-position filter wheel and cooled CCD;
`up to four dyes (500—660 nm in 32 X 5-nm
`detection of up to four dyes in a single tube;
`bins)
`detection range of 505-620 nm
`
`Proprietary intensifiertechnology; up to four
`targets and four fluorophores in a single
`tube; 400-700 nm
`
`Format/capacity
`
`384- or 96-well microplates; Micro Fluidic
`Card
`
`96-well microplates; 8-strip and individual
`tubes
`
`96-well plates; 0.2-mL tubes or strips of
`tubes
`
`Special features
`
`User-interchangeable blocks; automation
`accessory for unattended plate loading and
`unloading
`
`Two-fold resolution guaranteed
`
`NIST-traceable temperature performance;
`tvvo-fold resolution in starting material;
`customizable filter wheel
`
`tie Adams of Trudeau Institute’s Molec-
`
`ular Biology Core Facility, another rea-
`son that the technique’s popularity is
`rising is because it is versatile. It has be-
`come invaluable for validating microarray
`data; allelic discrimination assays, such as
`single-nucleotide polymorphism (SNP)
`detection; pathogen detection; and viral
`load measurements, to name a few.
`The explosion in gene-expression data
`coming from microarray experiments is
`
`PCR to quantitate gene expression ir1 dys-
`trophic tissue. “What I can do is look at
`the 100 or so genes .
`.
`. that look excit-
`ing from the chip, and I can go back to
`the real-time PCR on lots of additional
`
`samples at not much cost.”
`But the proof is always in the pud-
`ding, and until real-time PCR had accu-
`mulated a track record, researchers were
`skeptical of the technique, according to
`Ginzinger. “Anyone who had worked on
`
`”What I can do is look at the ‘I00 or so genes...
`
`that look exciting from the chip, and I can go
`
`back to the real-time PCR on lots of additional
`
`samples at not much cost."
`
`also fiieling interest, because researchers
`are finding that real-time PCR is a con-
`venient way to validate microarray re-
`sults. “I don’t think even now, real-time
`PCR can compete with chips that look at
`thousands of genes in one experiment. If
`I had to do all those genes in PCR, it’s
`not cost effective,” says Harvard Univer-
`sity’s Louis Kunkel, who uses real-time
`
`PCR knew that sometimes it worked;
`sometimes it didn’t. It was always a bit
`of a mystery,” he says. The difference
`is that the real-time PCR instruments
`
`provide a measure of control not pos-
`sible with a regular thermal cycler.
`“Now, with ways to monitor in real
`time, we can measure what we previ-
`ously cou.ldn’t measure.”
`
`176 A
`
`ANALYTICAL CHEMISTRY I APRIL 1, 2003
`
`The roots of real-time PCR
`
`Real-time PCR is not exactly new. Over
`a decade ago, scientists from Roche at-
`tached a fluorescence detector to a ther-
`
`mal cycler and demonstrated that PCR
`could be monitored by incorporating a
`fluorescent dye—in their case, ethidium
`bromide—into the reaction (1 The fluo-
`rescence intensified as the dye intercalated
`into the double-stranded PCR product.
`Soon after, dedicated instruments for
`conducting and monitoring the reaction
`in real time started to appear. Their pri-
`mary advantage was the ability to quan-
`titate the amount of starting material.
`But that wasn’t all. Because no post-re-
`action processing was necessary, this new
`method afforded time savings, and be-
`cause the reaction occurred in a closed
`
`tube, contarr1ination—a problem partic-
`ular to PCR—was less likely.
`The inherent variability of convention-
`al PCR, which traditionally looks only at
`the accumulated end product, confounds
`attempts to use it to measure the amount
`of a particular messenger RNA (mRNA)
`or to determine the number of copies
`of a gene. Because the reagents used in
`the reaction are eventually depleted or
`because the accumulation of a reaction
`
`product may inhibit fiirther amplification,
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`product review
`
`Table 1. Selected real—time PCR instruments (continued).
`
`Product
`
`Company
`
`Smartcycler ll
`
`RAPID
`
`Cepheid
`904 Caribbean Dr.
`Sunnyvale, CA 94089
`888-838-3222
`
`Idaho Technology, Inc.
`390 Wakara Way
`Salt Lake City, UT 84108
`800-735-6544
`
`URL
`
`wvvw.smartcycler.com
`
`wvvw.idahotech.com
`
`List price (U.S.D.l
`
`$31,800
`
`LEDs
`
`Excitation
`
`Detection
`
`$55,000
`
`LED at 475 nm
`
`Silicon photodetectors measure f|uo-
`rescent signal in four distinct channels
`
`Three-color optics: 530, 640, and
`705 nm
`
`Formatlcapacity
`
`16 sites; uses 25- or 100-uL po|ypro-
`pylene tubes
`
`32 slots; uses glass capillaries
`
`Configuring real-time
`measurements
`
`Special features
`
`16 sites can run independently (random Meets military specifications; field-
`portable; detector autocall software;
`access) or simultaneously; can link up
`melting curve analysis; 110- or 220-V
`to 6 processing blocks together for
`96-site capacity
`interchangeable
`
`were sent to a CCD camera for detecfion.
`
`Although the data was collected during
`the run—which is to say, in real time—it
`was displayed only at the end of the run.
`Today’s instruments collect and display
`the data (or have the option to do so) in
`real time. This adds another level of time
`
`savings, because researchers can spot bad
`reactions and abort or, in some machines,
`tweak the reaction conditions to improve
`the outcome.
`
`Since the introduction of Applied Biosys-
`tems’ ABI 7700 and Roche’s LightCy—
`cler, which followed on its heels, several
`companies have entered the market (as
`seen in Table 1). Current instruments
`use various configurations for exciting
`the fluorescent probes and collecting the
`resulting signals. Whereas the original
`machines excited the fluorophores with
`lasers—and ABI’s latest instrument, the
`7900HT, still does—most manufactur-
`ers have moved away from lasers to ei-
`ther light-emitting diodes (LEDs) or
`halogen/tungsten lamps. This helps re-
`duce the cost of the machine, allows for
`
`at some point, the typical PCR reaches a
`plateau that is unrelated to the amount
`of the starting material. Hence, the final
`amount of accumulated product is not
`informative.
`
`However, the rate at which the reac-
`tion proceeds before it reaches the plateau
`phase is informative, and it is directly
`proportional to the amount of initial
`target material. By collecting data as the
`reaction occurs—from the early phase
`when the product is undetectable and
`through the exponential amplification
`phase—it is possible to find the region
`of the curve where linearity exists; this
`is the so-called crossing threshold (CT),
`at which the amplification crosses over
`into exponential territory. Knowing the
`CT value lets you calculate the relative
`and, with the right standards, absolute
`amounts of starting material.
`The first detection scheme, with its
`generic DNA dye, was soon supplanted by
`more elegant and expensive fluorogenic
`probes, designed to hybridize to individ-
`ual genes. In the
`state, a fluorogenic
`probe is configured with a quencher in
`close proximity to the fluorescent dye on
`a sequence-specific probe. The two mol-
`ecules are separated when the probe hy-
`bridizes to its complementary sequence
`during the PCR, and the dye emits fluo-
`rescence in proportion to the reaction
`
`rate. This approach introduced a measure
`of specificity that was lacking with generic
`DNA dyes, which would bind to any dou-
`ble-stranded DNA in the tube, including
`pairs of primers hybridized to each other
`(“primer-dimers”) and to unwanted cross-
`reacting DNA fragments. Interestingly,
`although hybridization probes are still in
`
`Interestingly, although hybridization probes are
`
`still in wide use by those doing real—time PCR—
`
`with new designs coming out all the time—
`
`generic probes are coming back in fashion.
`
`wide use by those doing real—time PCR—
`with new designs coming out all the
`time—generic probes are coming back in
`fashion. One reason is that, as labs gear up
`to look at potentially hundreds of differ-
`ent mRNAs, preparing individual probes
`can be onerous as well as expensive.
`Applied Biosystems introduced the first
`real—time PCR instrument in 1997. This
`
`pioneering instrument, the ABI Prism
`7700, used a bank of fiber-optic cables to
`deliver light fiom a laser to the reaction
`tubes and to collect fluorescent signals
`fi'om each tube periodically; these signals
`
`a smaller footprint, and increases the
`range of wavelengths used in the excita-
`tion, thereby affording greater flexibility
`in the choice of dyes.
`Signal collection, too, is done in vari-
`ous ways. Some machines read all samples
`simultaneously with a CCD camera or
`similar technology, whereas others read
`the samples individually and sequentially
`with scanning optics. Reading the whole
`plate of reactions with a CCD camera is
`the simpler and cheaper approach, and
`for many applications that don’t require a
`great deal of precision, it is perfectly ade-
`
`APRIL 1. 2003 / ANALYTICAL CHEMISTRY
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`177A
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`
`product review
`
`Table 1. Selected real-time PCR instruments (continued).
`
`Product
`
`Company
`
`DNA Engine Opticon and Opticon 2
`
`Rotor-Gene 3000
`
`Light Cycler
`
`MJ Research, Inc.
`590 Lincoln St.
`Waltham, MA 02451
`388-PELTIER
`
`Phenix Research Products
`3540 Arden Rd.
`Hayward, CA 94545
`800-767-0665
`
`Roche Applied Science
`9115 Hague Rd.
`P.0. Box 50414
`Indianapolis, IN 46250
`800-262-491 1
`
`URL
`
`wvvw.mjr.com
`
`vwvw.phenix1.com
`
`wvvw.roche-applied-science.com
`
`List price (U.S.D.I
`
`$23,590 to upgrade existing instrument or
`$29,980 for an integrated system
`
`$39,990 (unlimited seats)
`
`High-power LEDs at 470, 530, 585, and
`Sequential excitation from arrayed LEDs;
`Opticon: 450-495 nm; Opticon ll: 470-505 nm 625 nm
`
`$57,500
`
`Blue LED
`
`Excitation
`
`Detection
`
`Photomultiplier tube; Opticon: 515-545 nm;
`Opticon ll: 523-543 and 540-700 nm
`
`510-, 555-, 610-, and 660-nm bandpass;
`585- and 610-nm highpass
`
`Three-channel detection at 530, 640, and
`710 nm
`
`Format/capacity
`
`96-well microplates; 0.2-mL tubes or strips
`of tubes
`
`Thirty-six 0.2-mL tubes; seventy-two 0.1-mL
`strip tubes
`
`32 glass capillaries
`
`Special features
`
`Accessory to DNA Engine cycler; optical
`scanning system minimizes cross-talk be-
`tween wells; can run gradients across 96-
`well block; software also does melt-curve
`and genotyping analyses
`
`Air- and centrifuge-based heating and
`cooling; temperature uniformity of 0.01 °C
`across all samples; no need for passive
`reference
`
`Melting curve analysis; automatically deter-
`mines starting concentration when a stan-
`dard curve is included; optimization of fluo-
`rescence in each position
`
`quate. On the other hand, sampling the
`reactions individually may provide better
`accuracy and precision than working with
`a snapshot of the entire plate of reactions.
`Unfortunately, the moving parts needed
`for scanning can cause optical Variation,
`says Ginzinger.
`Instrument manufacturers have differ-
`
`ent ways of handling optical variability.
`The ABI instruments, for example, read a
`reference sample that has been spiked into
`
`Phenix Research’s Rotor-Gene, which
`centrifuges the samples at low speed
`during the entire PCR, moves the sain-
`ples through the detector, rather than
`the other way around. MI Research has
`devised an optical scanner with no mov-
`ing parts for its instrument.
`Collecting individual signals also re-
`duces cross talk between samples, which
`can be especially problematic when a
`sample with a low signal sits next to one
`
`Most of today's real-time PCR instruments
`
`use a 96-well format, which product managers
`
`say gives adequate throughput for most of
`
`their customers.
`
`each reaction, called a passive reference,
`which the software uses to adjust for varia-
`tions. Other manufacturers recommend
`
`periodically reading a reference plate,
`which maps out Variations in the optical
`path and thermal variation in the PCR
`plate. And some instruments have com-
`pletely novel solutions. For example,
`
`with a high signal. Eliminating cross talk
`increases the dynamic range, according
`to Mike Mortillaro, MI Research’s vice
`president of sales and marketing. For ex-
`ample, Mortillaro reports that M] Re-
`search’s Opticon, which samples individ-
`ual reactions, has a dynamic range of 10
`orders of magnitude, compared with a
`
`175 A
`
`ANALYTICAL CHEMISTRY I APRIL 1, 2003
`
`typical dynamic range of 5-6 when the
`entire microwell plate is read at once.
`
`Faster yet
`Throughput is the latest buzzword in
`genome centers and molecular biology
`labs, where the gene-by- gene approach
`has been supplanted by modern tech-
`nologies that interrogate thousands of
`genes in a single experiment. And these
`technologies—n1icroarray experiments,
`SNP detection, and genotyping assays—
`are increasing researchers’ appetites for
`high-throughput real-time PCR.
`Most of today’s real-time PCR instru-
`ments use a 96-well format, which prod-
`uct managers say gives adequate through-
`put for most of their customers; a typical
`application would be picking out genes
`by the hundreds from an earlier expres-
`sion profile experiment using rnicroarrays.
`However, for those really high-through-
`put applications, Applied Biosystems has
`come out with the ABI Prism 7900HT,
`which is the first system that can handle
`384-well plates. To speed things up even
`further, the 7900 can be used with lab
`robots for automation.
`
`Another way of increasing through-
`put is by multiplexing—performing two,
`
`THERMO FISHER EX. 101 1
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`

`
`product review
`
`according to Idaho Technologies’ Matt
`Scullion. The SmartCycler has 16 sepa-
`rate reaction chambers that can each be
`
`run independently, allowing different
`protocols to be run simultaneously.
`Norman Schaad, of the USDA’s For-
`eign Disease Research Unit, finds this
`unique aspect of the SmartCycler usefiil,
`because plants are plagued by hundreds
`of diseases. Using portable real-time PCR
`machines in the field allows him to assay
`for several diseases at once. “If I can ex-
`
`tract bacteria from a plant and do a di-
`rect PCR reaction in less than an hour,
`I don’t know how it could be done any
`better or easier,” he says.
`
`Peering into the future
`With nine instruments on the market and
`
`more to come (Techne and Idaho Tech-
`nologies will be launching new real-time
`PCR machines this year), real-time PCR
`researchers have choices like never before.
`
`So what else do suppliers need to do?
`Reducing the reaction volume will be
`the next major advance. Performing reac-
`tions on the nanoliter scale would make a
`
`world of difference, Ginzinger says, when
`the amount of material is limited, as in
`cancer research. And the field may experi-
`ence even more growth if high-through-
`put applications like genotyping and SNP
`detection migrate to real-time PCR
`
`Laura DeFrancesco is a freelance writer based
`
`in Pasadena, Calif.
`
`References
`(1)
`Higuchi, R.; Fockler, C.; Dollinger, (3.; Watson,
`R. Biotechnolagy1993. 9, 1026-1030.
`Raja, S.; El-Hefnawy, T.; Kelly, L. A.;
`Chestney, M. L.; Luketich, J. D.; Godfrey, T. E.
`Clin. Chem. 2002. 8, 1329-1337.
`
`(2)
`
`Upcoming product reviews
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`August 1: Surfuce ucoustic wuve devices
`September 1: Luborutory workstations
`umi robotic:
`
`October 1: Ion mobility mun‘ spectrometer:
`November 1: Prepurutive liquid
`cbromutogrupbx
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`If your company manufactures any of
`these products, please contact us at
`analytical@acs.org.
`
`three, or even four PCRs simultaneously
`in a single tube. In addition to increas-
`ing throughput, multiplexing reduces
`costs by saving on reagents and uses less
`target material, which can be important
`when samples are limited. Recognizing
`the potential benefits of multiplexing,
`several manufacturers such as Bio-Rad
`
`and Stratagene have incorporated fea-
`tures into their instruments that allow
`
`for detecting and separating the signals
`from multiple dyes.
`However, according to the experts,
`multiplexing poses challenges besides
`data collection—particularly in designing
`and optimizing the reactions. Reactions
`that are optimized singly need to be re-
`optirnized when multiplexed. Primers
`also have to be carefully designed to
`avoid regions of possible cross—reaction
`and competition among the target se-
`quences. And, even with well—designed
`primers, an efficient reaction could im-
`pinge on other, less-efficient ones by
`causing product inhibition or by deplet-
`ing the reagents. Unless you are doing
`the same three or four reactions on nu-
`
`merous samples, the consensus among
`real-time PCR users is that multiplexing
`is not worth the investment of time, es-
`pecially considering the speed with which
`
`“It’s bet-
`single reactions can be run
`ter to run ‘single-plexes’ and move onto
`the next reaction,” says Ginzinger.
`However, in situations such as clinical
`diagnosis, multiplexing may be critical.
`With reactions finishing in less than an
`hour, researchers are gearing up to use
`real-time PCR for analyzing surgical sam-
`ples while the patient is still on the table—
`perhaps to check the margins of a tumor
`being removed. But to do it right requires
`reading a minimum of three dyes per sam-
`ple: the gene of interest, a positive con-
`trol, and a negative control. You would
`want to be certain of any negative result
`to ensure that sample preparation errors
`were not the cause.
`
`Outstanding in the field
`In part because of current concerns over
`bioterrorism, real-time PCR machines
`are finding their way into the field. The
`speed with which pathogens can be de-
`tected and the ability to detect mutant
`forms make this technique attractive for
`field applications. Two portable ma-
`chines, Idaho Technologies’ RAPID and
`Cepheid’s SmartCycler, are taking the
`lead here. The RAPID meets military
`specifications and is rugged enough to
`survive a one-meter drop to pavement,
`
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