Real-Time PCR Takes Center Stage

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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

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ntil recently, the real-time polymerase 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 superior to most any other technique, such as Northern blots, dot blots, or RNAse protection assays. Unfortunately, though, the cost of the instruments and the designer 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 Francisco’s Genome Analysis Core Facility, “In the interim of only a couple of years, there was a 10-fold increase in the number 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 sequencing or high-density microarrays, both of which are commonplace in core facilities and in many individual investigator 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 comprehensive but to indicate the types of options available to potential buyers.

Quantitating nucleic acids was the initial application and may still be what researchers most often use it for, but that’s not the whole story. According to ScotA P R I L 1 , 2 0 0 3 / A N A LY T I C A L C H E M I S T R Y

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product review

Table 1. Selected real-time PCR instruments. Product

ABI PRISM 7900HT

ABI PRISM 7000

iCycler iQ

Company

Applied Biosystems 850 Lincoln Centre Dr. Foster City, CA 94404 800-327-3002

Applied Biosystems 850 Lincoln Centre Dr. Foster City, CA 94404 800-327-3002

Bio-Rad Laboratories 2000 Alfred Nobel Dr. Hercules, CA 94547 800-4-BIORAD

URL

www.appliedbiosystems.com

www.appliedbiosystems.com

www.bio-rad.com/iCycler

List price (U.S.D.)

$90,000; $130,000 with optional automation accessory

$47,250

$49,500

Excitation

Extended-life argon-ion laser and dual-axis synchronous scanning head

Tungsten/halogen lamp

Tungsten/halogen lamp with five-position filter wheel

Detection

Spectrograph and cooled CCD; detection of Four-position filter wheel and cooled CCD; Proprietary intensifier technology; up to four detection of up to four dyes in a single tube; targets and four fluorophores in a single up to four dyes (500–660 nm in 32  5-nm detection range of 505–620 nm bins) tube; 400–700 nm

Format /capacity

384- or 96-well microplates; Micro Fluidic Card

Special features

Two-fold resolution guaranteed User-interchangeable blocks; automation accessory for unattended plate loading and unloading

tie Adams of Trudeau Institute’s Molecular Biology Core Facility, another reason that the technique’s popularity is rising is because it is versatile. It has become 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

96-well microplates; 8-strip and individual tubes

PCR to quantitate gene expression in dystrophic tissue. “What I can do is look at the 100 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.” But the proof is always in the pudding, and until real-time PCR had accumulated 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 100 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 fueling interest, because researchers are finding that real-time PCR is a convenient way to validate microarray results. “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 University’s Louis Kunkel, who uses real-time 176 A

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 possible with a regular thermal cycler. “Now, with ways to monitor in real time, we can measure what we previously couldn’t measure.”

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96-well plates; 0.2-mL tubes or strips of tubes NIST-traceable temperature performance; two-fold resolution in starting material; customizable filter wheel

The roots of real-time PCR Real-time PCR is not exactly new. Over a decade ago, scientists from Roche attached a fluorescence detector to a thermal cycler and demonstrated that PCR could be monitored by incorporating a fluorescent dye—in their case, ethidium bromide—into the reaction (1). The fluorescence 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 primary advantage was the ability to quantitate the amount of starting material. But that wasn’t all. Because no post-reaction processing was necessary, this new method afforded time savings, and because the reaction occurred in a closed tube, contamination—a problem particular to PCR—was less likely. The inherent variability of conventional 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 further amplification,

product review

Table 1. Selected real-time PCR instruments (continued). Product

SmartCycler II

RAPID

Company

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

www.smartcycler.com

www.idahotech.com

List price (U.S.D.)

$31,800

$55,000

Excitation

LEDs

LED at 475 nm

Detection

Silicon photodetectors measure fluoThree-color optics: 530, 640, and rescent signal in four distinct channels 705 nm

Format/capacity

16 sites; uses 25- or 100-µL polypropylene tubes

32 slots; uses glass capillaries

Special features

16 sites can run independently (random access) or simultaneously; can link up to 6 processing blocks together for 96-site capacity

Meets military specifications; fieldportable; detector autocall software; melting curve analysis; 110- or 220-V interchangeable

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 reaction 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 individual genes. In the initial state, a fluorogenic probe is configured with a quencher in close proximity to the fluorescent dye on a sequence-specific probe. The two molecules are separated when the probe hybridizes to its complementary sequence during the PCR, and the dye emits fluorescence in proportion to the reaction

were sent to a CCD camera for detection. 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.

Configuring real-time measurements

rate. This approach introduced a measure of specificity that was lacking with generic DNA dyes, which would bind to any double-stranded DNA in the tube, including pairs of primers hybridized to each other (“primer-dimers”) and to unwanted crossreacting DNA fragments. Interestingly, although hybridization probes are still in

Since the introduction of Applied Biosystems’ ABI 7700 and Roche’s LightCycler, 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 manufacturers have moved away from lasers to either light-emitting diodes (LEDs) or halogen/tungsten lamps. This helps reduce the cost of the machine, allows for

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 different 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 from a laser to the reaction tubes and to collect fluorescent signals from each tube periodically; these signals

a smaller footprint, and increases the range of wavelengths used in the excitation, thereby affording greater flexibility in the choice of dyes. Signal collection, too, is done in various 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-

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product review

Table 1. Selected real-time PCR instruments (continued). Product

DNA Engine Opticon and Opticon 2

Rotor-Gene 3000

Light Cycler

Company

MJ Research, Inc. 590 Lincoln St. Waltham, MA 02451 888-PELTIER

Phenix Research Products 3540 Arden Rd. Hayward, CA 94545 800-767-0665

Roche Applied Science 9115 Hague Rd. P.O. Box 50414 Indianapolis, IN 46250 800-262-4911

URL

www.mjr.com

www.phenix1.com

www.roche-applied-science.com

List price (U.S.D.)

$23,590 to upgrade existing instrument or $29,980 for an integrated system

$39,990 (unlimited seats)

$57,500

Excitation

High-power LEDs at 470, 530, 585, and Sequential excitation from arrayed LEDs; Opticon: 450–495 nm; Opticon II: 470–505 nm 625 nm

Detection

Photomultiplier tube; Opticon: 515–545 nm; Opticon II: 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 between wells; can run gradients across 96well 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 determines starting concentration when a standard curve is included; optimization of fluorescence 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 different 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 samples through the detector, rather than the other way around. MJ Research has devised an optical scanner with no moving parts for its instrument. Collecting individual signals also reduces 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 variations. 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 completely novel solutions. For example, 178 A

with a high signal. Eliminating cross talk increases the dynamic range, according to Mike Mortillaro, MJ Research’s vice president of sales and marketing. For example, Mortillaro reports that MJ Research’s Opticon, which samples individual reactions, has a dynamic range of 10 orders of magnitude, compared with a

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Blue LED

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 technologies that interrogate thousands of genes in a single experiment. And these technologies—microarray experiments, SNP detection, and genotyping assays— are increasing researchers’ appetites for high-throughput real-time PCR. 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; a typical application would be picking out genes by the hundreds from an earlier expression profile experiment using microarrays. However, for those really high-throughput 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 throughput is by multiplexing—performing two,

product review

Table 1. Selected real-time PCR instruments (continued). Product

MX 4000

Quantica

Company

Stratagene 11011 N. Torrey Pines Rd. La Jolla, CA 92037 800-894-1304, ext. 2

Techne, Inc. 743 Alexander Rd. Princeton, NJ 08540 800-225-9243

URL

www.stratagene.com

www.techneusa.com

List price (U.S.D.)

$59,995

Excitation

Tungsten/halogen bulb at 350–750 nm Halogen white light Scanning fiber optics; four optical channels allow detection of up to four dyes (350–830 nm)

Detection

96-well format (plates, tubes, or strips)

Format/capacity Special features

Versatile software package; wide range of chemistry and dye choices; four-dye multiplexing; two-fold resolution in starting material

three, or even four PCRs simultaneously in a single tube. In addition to increasing 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 features 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 reoptimized when multiplexed. Primers also have to be carefully designed to avoid regions of possible cross-reaction and competition among the target sequences. And, even with well-designed primers, an efficient reaction could impinge on other, less-efficient ones by causing product inhibition or by depleting the reagents. Unless you are doing the same three or four reactions on numerous samples, the consensus among real-time PCR users is that multiplexing is not worth the investment of time, especially considering the speed with which

Photomultiplier tube; multiple fluorescence

according to Idaho Technologies’ Matt Scullion. The SmartCycler has 16 separate reaction chambers that can each be run independently, allowing different protocols to be run simultaneously. Norman Schaad, of the USDA’s Foreign Disease Research Unit, finds this unique aspect of the SmartCycler useful, 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 extract bacteria from a plant and do a direct PCR reaction in less than an hour, I don’t know how it could be done any better or easier,” he says.

96-well format

Peering into the future

Wizard-based software; multiplexing capability; open-format system, allowing end users to decide the chemistry and 96-well plates of their own choice

With nine instruments on the market and more to come (Techne and Idaho Technologies 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 reactions 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 experience even more growth if high-throughput applications like genotyping and SNP detection migrate to real-time PCR.

single reactions can be run (2). “It’s better 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 samples 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 sample: the gene of interest, a positive control, and a negative control. You would want to be certain of any negative result to ensure that sample preparation errors were not the cause.

Laura DeFrancesco is a freelance writer based in Pasadena, Calif.

References (1) (2)

Higuchi, R.; Fockler, C.; Dollinger, G.; Watson, R. Biotechnology 1993, 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.

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 detected and the ability to detect mutant forms make this technique attractive for field applications. Two portable machines, 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,

Upcoming product reviews July 1: Hybrid mass spectrometry systems August 1: Surface acoustic wave devices September 1: Laboratory workstations and robotics October 1: Ion mobility mass spectrometers November 1: Preparative liquid chromatographs If your company manufactures any of these products, please contact us at [email protected].

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