Realtime PCR instrumentation an overview

It would be impractical to present technical information for every real-time instrument on the market today. However, it is important that users of realtime instruments have a basic understanding of how their instruments work and have some understanding of their physical capabilities and limitations. There are three major components in any real-time instrument: 1) the light source, which determines the range of reporter dyes the instrument is capable of using; 2) the detection system, where the spectral range and sensitivity of any assay will be determined; and 3) the thermocycling mechanism, the determinant of the speed at which an assay can be run, the uniformity of the temperature changes from sample to sample and the number of samples that can be accommodated at any one time.

There are currently four different light sources available in real-time instruments, argon-ion laser; LED (light emitting diode) lasers, quartz halogen tungsten lamps and xenon lamps. Each of these different light sources will determine the capabilities of the instruments that use them. The argon-ion laser, now available solely in the ABI 7900, emits powerful light primarily at 488 nm. For reporters such as 6-FAM, this is an ideal excitation wavelength. However, as you move toward the red spectrum, the excitation energy from a 488 nM light source will result in a weaker stimulation of a reporter dye with maximal excitation above 500 nM with a concomitant weaker emission signal. Weaker signals can limit the utility of a reporter dye. The ABI 7900 captures the emitted reporter signal using a CCD (charge-coupled device) camera and relies on software to parse out multiple emitted wavelengths. The ABI 7900 utilizes a Peltier-based thermo-cycler. There are currently 4-exchangable thermocycler blocks available for 96- and 384-well plates.

A single LED laser can be found in the LightCycler® 1 and LightCycler® 2 from Roche. Unlike classic lasers, LED lasers emit light within a 30-40 nM spectral range. The energy output of these lasers is not as strong as the argon-ion lasers but their energy use and generated heat are significantly less with a lower cost per unit. Both LightCycler®, 1 and 2, use a single blue LED laser as an excitation source. The LightCycler® have a number of photodetection diodes, each of which is specific for an increasing and narrow wavelength range of light. Unlike the plates used in the block-based ABI 7900, the LightCycler® uses glass capillary tube reaction vessels with a high surface to volume ratio. Coupled with the hot air driven thermocycler system, these machines can complete a 40 cycle experiment in about 30 minutes compared to thermocycler/plate-based instruments times of approximately 2 hours.

Currently, the most used light source for new instruments is a quartz tungsten halogen lamp. These lamps are capable of emitting a steady beam of light from 360 nM to over 1,000 nM. They are sometimes referred to as 'white light' sources as they emit light over the entire visible spectrum. Unlike either of the laser light sources described above, two sets of excitation and emission filters are required to select the desired wavelengths for multiple reporter dyes. The number of filters available determines the level of multiplexing the instrument can support. Some of these instruments use a PMT (photomultiplier tube) and others a CCD camera for signal detection depending upon how the light is captured from the plate.

One of the newest instruments coming onto the market, the Roche LightCycler® 480, has a xenon lamp, which has a higher light intensity compared to the quartz tungsten-halogen lamp and covers a similar spectral range. This machine will have 5 excitation filters coupled to 6 emission filters which will potentially allow 6 different assays in a single, multiplexed reaction. This instrument will support both 96- and 384-well plates in a Peltier-based thermocycler that is reported to have the most uniform thermal characteristics of any instrument.

Table 1.4 provides a selected overview of some of the most important features for the newest instruments available from the major real-time PCR instrument vendors. The ABI 7700, the original real-time instrument, is also listed for comparison.

Besides the hardware itself, the next most important component of a

Table 1.3 Dyes available for use in real-time PCR

Free dyes

Max. ab (nM)

Max. em (nM)

SYBR® Green I

497

525

EvaGreen™

497

525

BOXTO™

515

552

Reporter dyes

Max. ab (nM)

Max. em (nM)

Pulsar® 650

460

650

Fluorescein™

492

520

6-FAM

494

518

Alexa 488™

495

519

JOE™

520

548

TET™

521

536

Cal Fluor Gold 540™

522

544

Yakima Yellow™

530

549

HEX™

535

556

Cal Fluor Orange 560™

538

559

VIC™

538

554

Quasar® 570

548

566

Cy3™

552

570

TAMRA™

565

580

Cal Fluor Red 590™

569

591

Redmond Red™

579

595

ROX™

580

605

Cal Fluor Red 635™

618

637

LightCycler®640

625

640

Cy5™

643

667

Quasar® 670

647

667

LightCycler®705

685

705

Dark dyes

Max. ab (nM)

Max. em (nM)

DABCYL

453

None

BHQ0™

495

None

Eclipse™

522

None

Iowa Black™ FQ

531

None

BHQ1™

534

None

BHQ2™

579

None

Iowa Black™ RQ

656

None

BHQ3™

680

None

Pulsar, Cal Fluor, BHQ, Quasar and Pulsar are registered trademarks of Biosearch Technologies; Alexa is a registered trademark of Molecular Probes/Invitrogen; Iowa Black is a registered trademark of IDT; Redmond Red, Yakima Yellow and Eclipse are registered trademarks of Nanogen; LightCycler®640 and LightCycler®705 are registered trademarks of Roche; Evagreen is the registerd trademark of Biotium, Inc.

Pulsar, Cal Fluor, BHQ, Quasar and Pulsar are registered trademarks of Biosearch Technologies; Alexa is a registered trademark of Molecular Probes/Invitrogen; Iowa Black is a registered trademark of IDT; Redmond Red, Yakima Yellow and Eclipse are registered trademarks of Nanogen; LightCycler®640 and LightCycler®705 are registered trademarks of Roche; Evagreen is the registerd trademark of Biotium, Inc.

real-time instrument is the software package. This aspect of any real-time instrument should not be overlooked. An instrument with good hardware but a poor software package can diminish the overall user experience. Although many software packages offer different levels of automated data analysis, it should be pointed out that the results may not be as good as manual manipulation of the baseline and threshold settings by the investigator (see Chapter 2). Most programs offer ways to export the metadata into a file that can be opened in Microsoft Excel® and the graphical views into one or more common graphical formats. These exports can be very useful for more extensive data analysis in other programs and for incorporating data or graphics in either publications or presentations (see Chapters 2 and 3). For these reasons, it is important that the user explore all aspects of the software to ensure they are getting the most out of the data obtained by their real-time instruments.

The trends in real-time PCR instrumentation development have been in three directions. First has been an evolution in fluorescent dye detection. In the early years of real-time PCR, assays were run with one assay per reaction. This was primarily because the hardware was geared to the excitation of 6-FAM as a reporter. As more and more instruments came onto the market with more extensive capabilities in their spectral sophistication, it has become possible to excite a larger spectrum of reporters. Today, many instruments offer 5 or 6 unique excitation/emission filter combinations. In theory, this would allow up to 5 or 6 transcript or gene assays to be measured within a single multiplexed reaction. The emitted light from each reporter dye is physically isolated from the others by the filters as it is collected. This minimizes spectral spill over which was a real problem with the early instruments that relied entirely on software to differentiate signals from different reporter dyes. Thus, the instrumentation is now equipped for extensive assay multiplexing. The chemistry is just now starting to catch up and will be discussed in the next section.

The second development in real-time instrumentation has been towards faster cycling capabilities. For the Roche LightCycler® 1 and 2 and the Corbett Rotorgene, fast cycling has always been a feature because samples were not heated in a block thermocycler. However, these machines offered less than 96 sample capacities per run cycle (Table 1.4). The manufacturers of instruments with block thermocyclers are now offering faster cycling times coupled to new assay chemistries. This trend will undoubtedly reduce the time it takes to run each plate. A reduction in run times becomes more critical for high throughput users and will most likely lead to further reductions in run times.

The third development is the increase in sample throughput. For some time now, the ABI 7900 has been the only real-time instrument on the market capable of running 384-well plates. With the announcement of the new Roche LightCycler® 480, that will no longer be the case. The LightCycler® 480 will have the added advantage of offering true multiplexing capabilities for up to 6 dyes in each reaction. Although 384-well plates may seem overkill for the individual laboratory, for the pharmaceutical industry, even 384-well plates cannot provide the desired throughput. For true high throughput screening, 1,536-well plates are now the standard. For research core laboratories that utilize liquid handling robots to set up the reactions, however, 384-well plates are well positioned to raise the throughput bar over individual laboratories where 96-well plates have become the upper limit for hand pipetting.

The combination of multiplexed reactions, faster cycling times and higher sample throughputs per run through the use of robotics mean that the throughput potential of real-time PCR has been amplified at least 40fold from the original instruments. What the throughput upper limit for real-time instrumentation will be is not clear at this time. What is clear is that limit has not yet been achieved.

Table 1.4 A comparison of features for the most commonly used real-time instruments

Instrument/ configuration

ABI Prism® 7700

ABI Prism® 7500

ABI Prism® 7900

Bio-Rad iQ®5

Bio-Rad MJR Chromo 4™

Light source

Argon laser

Tungsten-halogen

Argon laser

Tungsten-halogen

4 LEDs

Detector

CCD camera

CCD camera

CCD camera

CCD camera

4 photodiode detectors

Scanning system

96 fiber optic cables

Whole plate

Scanning head by row

Whole plate

Scanning photonics shuttle

Excitation wavelengths (s)

488 nm

450-650 nm

488 nm

400-700 nm

450-650 nm

Emission wavelengths

500-650 nm

500-700 nm

500-660 nm

400-700 nm

515-730 nm

Multiplex capability (number)

2

5

2

5

4

Sample vessel

96-well plate

96-well plate

96/384-well plates

96-well plate

96-well plate

Sample volume

25-100 pl

20-100 pl

20-100 |jl/96 5-20 jl/384

10-50 pl

20-100 pl

Thermocycler

Thermocycler

Sensitivity

9-logs

9-logs

9-logs

6-logs

10-logs

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