product review
DNA sequencers rely on CE With the newest systems, half a million bases can be read in a day. James P. Smith and Vicki Hinson-Smith
he Human Genome Project (HGP) formally began in October 1990 and was scheduled to last 15 years. Nevertheless, a draft of the genome was published this year, and many analytical chemists may have dislocated their shoulders patting themselves on the back. They should be proud. The HGP was completed years ahead of schedule and under budget, mainly because of automated, high-throughput capillary electrophoresis (CE) DNA sequencers— instruments that didn’t even exist in 1990. “CE has had a major impact on genomics,” said Barry Krager, director of the Barnett Institute for Chemical and Biological Analysis at Northeastern University. “When the human genome project began, investigators explored a number of techniques. Many thought that electrophoresis [EF] would not be the way to do it. They thought that some new technologies would be required because there were 3 billion bases to map, and EF was too slow. Over the years, however, the new experimental technologies couldn’t ‘cut the mustard.’ EF, in the meantime, went into a capillary format; then came replaceable separation matrixes and new automated instrumentation.” Aran Paulus of the Novartis Research Foundation has seen the same development. “The productivity of the new CE instrumentation has increased by a factor of 10 over what was available before. The slab-gel systems had up to 96 lanes, but you could only operate them maybe twice per day. Now, we have an automated operation around the clock. You
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need only place a 96-well sample plate in the instrument and walk away. Runs are complete in 2–3 hours,” he says.
“Today, with 100 of the new CE instruments, it would take only two months to sequence the human genome one
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product review
Table 1. Selected features of capillary electrophoresis DNA sequencers. Product
M egaBACE 500 and 1000
Prism 3100 and 3700
SCE 2410 and 9610
CEQ 2000XL
Com pany
Amersham Pharmacia Biotech, Inc. 800 Centennial Ave. Piscataway, NJ 08855-1327 732-457-8000 fax 732-457-0557
Applied Biosystems, Inc. 850 Lincoln Centre Dr. Foster City, CA 94404 650-570-6887 fax 650-570-2743
SpectruMedix Corp. 2124 Old Gatesburg Rd. State College, PA 16803 814-867-8600 fax 814-867-4513
Beckman Coulter, Inc. 4300 N. Harbor Blvd. Fullerton, CA 92835 714-773-8885 fax 714-773-8283
URL
www.megabace.com
www.appliedbiosystems.com
www.spectrumedix.com
www.beckmancoulter.com/ DNAnews
Num berof capillaries
MegaBACE 500: 48; MegaBACE 1000: 96
Prism 3100: 16 (uncoated); Prism 3700: 96 (uncoated)
SCE 2410: 24 (uncoated); SCE 9610: 96 (uncoated)
8, 16, 24, 32 (1–4 systems ganged) provide variable throughput; coated
Listprice (USD)
Price upon request
Prism 3100: $130,000; Prism 3700: $300,000
SCE 2410: $135,000; SCE 9610: $250,000
$85,000
Prism 3100: 192; Prism 3700: 1152 with POP-37 matrix
SCE 2410: 288; SCE 9610: 1152
224 per 8-capillary system
24-h sequenc- MegaBACE 500: 576; MegaBACE 1000: 1152 ing sam ple throughput, reading >500 bases Dim ensions H3W 3D
32" 3 40.7" 3 34.4"
Information not available
SCE 2410: 24" 3 20" 3 36"; SCE 9610: 28" 3 26" 3 43"
37" 3 24" 3 24"
Readlength
Up to 750, Phred 20
>900 at Phred 20 have been observed by customers
Up to 850 base pairs at Phred 20
700 bases (pUC-18) in 100 min (cycle time) at 98.5% accuracy
Separation m atrix
Proprietary linear polyacrylamide
Flowable noncrosslinked separation polymers: POP-5, POP-6, and POP-37
Proprietary LPA-based formulation Proprietary LPA-1 in a cartridge
Excitation laser
488/532 nm
Argon multiline laser
488/514 nm
Detection
Scanning confocal microscope with filters and four lensed photomultiplier tubes
Prism 3100: simultaneous dual side On-column CCD camera and spec- Filter wheel with 4 filters and illumination and in-capillary detec- trometer measures all capillaries a photomultiplier, 4 near-IR wavelengths tion; CCD detector measures all 16 simultaneously capillaries simultaneously. Prism 3700: sheath-flow detection with scanning laser; fluorescent light passes through a spectrograph, which images the light onto a CCD
Colors detect- 4 (different filter sets allow different dye sets to be used) ed perlane
5
2 diode lasers, 650 and 750 nm
Up to 10
4; WellRED dyes: D1, D2, D3, D4 (IR dyes) Dye terminator cycle sequencing, DNA fragment sizing and analysis
Applications
Sequencing, microsatellite, SNPs, Sequencing, fragment analysis, genotyping genotyping, mutation, and SNPs
Sequencing, fragment analysis, mutation discovery, forensics, SNP genotyping
Softw are
Sequence Analyzer, basecallers, Genetic Profiler, SNP Profiler, ScoreCard, Genetic Profiler Scorecard
BioLIMS, GenScan Sequencing GenMapper, SQL*GT, Genotyper
All software elements integrated Sequencing, genotyping, forensics, mutation screening, fragment into one package; sequence analysis; quality assessment; hetanalysis erozygote detection; confirmatory sequencing; fragment sizing; allele calling; and export
Special features
Higher precision for genotyping, longer readlength matrix; ThermoSequenase II and ET terminator chemistries; spectral separation on the fly; automatic “sleep” after the last run; upgrade path from MegaBACE 500 to 1000; easy to switch between applications
Supports both sequencing and fragment analysis applications; easy to switch between capillary lengths, matrixes, and chemistry kits; variable operating temperature: 18–65 °C; 3700 supports 96and 384-well plates
Full “walk-away” automation; direct sample injection from prepared tray; no transfer via robotics; capillaries housed in cartridge permit changeover to new array within 15 min; current monitoring provides capillary performance diagnosis; temperature-controlled capillary cartridge permits temperature ramping
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Capillary arrays are coated to reduce electroosmotic flow; samples automatically heat-denatured before introduction; sample preparation may be automated with the Biomek 2000 or FX nucleic acid workstations
product review
time through,” claims Paulus. “A few years ago, the top throughput available was about 25,000 bases per day, per instrument. Today, CE sequencing has raised this to more than 500,000.” The last product review on electrophoresis DNA sequencing was published five year ago (Anal. Chem. 1996, 68, 493 A–497 A); but at that time, highthroughput EF was based on slab-gel technologies. Although this technology is still used, CE now defines DNA sequencing. This review focuses on automated CE DNA sequencing systems. Table 1 summarizes the features of seven instruments marketed by four companies. The HGP was completed using instruments produced by two of these companies: Applied Biosystems, Inc. (ABI) and Molecular Dynamics, which was recently acquired by Amersham Pharmacia Biotech Inc. (APBiotech). ABI offers 16-capillary (Model 3100) and 96-capillary (Model 3700) systems. APBiotech sells an instrument called MegaBACE in both a 48- and a 96-capillary format. Other suppliers are Beckman Coulter with an 8-capillary instrument and Spectrumedix with 24- and 96-capillary instruments. These companies produce other DNA sequencing systems, ranging from single capillary to 384-capillary systems. Now that the HGP is complete, many investigators are changing their research emphasis from sequencing to related studies aimed at applying genomics, so instrument companies are designing for multiple applications.
Separations Capillary instruments have significant advantages over slab-gel systems, say the experts, because reading slab-gel systems requires time-consuming postrun tracking to find fragments, whereas data collection with capillaries is always in the same location. Moreover, the newer instruments automatically inject the CE separation matrix into all the capillaries at once before loading the samples; samples are then loaded onto the capillaries by electrokinetic injection. Sample preparation is the same for both CE and slab-gel systems. The key aspect is that, depending on the terminal DNA base, one of four different dyes is
attached to various DNA fragments. Thus, the sample contains many fragments, each tagged by one of the dyes. Because lower molecular weight fragments move faster through the capillary, the DNA sequence can be determined by the dye color sequence of the eluting fragments. The larger molecular weight fragments, which have been on the column for the longest time, are less well resolved and therefore more difficult to identify. The capillaries have i.d.s of 35–75 µm and are 30–60 cm in length, depending on the desired readlength, which is the number of DNA bases that can be accurately determined in a single run on a single column. Readlengths are typically in the range of 500–700 bases, but some instrument manufacturers boast readlengths as high as 1000. Instruments with 96 capillaries commonly have run times of 2–3 h; therefore, if all goes well, more than half a million bases can be read per day. The readlength of a CE instrument is an important performance parameter, which is specified in different ways. The typical readlength depends on factors such as the quality of the sample preparation, DNA and impurity concentrations, and instrumental parameters. The optimization of readlengths for a particular application involves trade-offs between the run duration, capillary length, type of separation matrix, temperature, and electrophoretic voltage. The specified readlength is based on the sequencing of a known standard under specific conditions. If all manufacturers used the same standard procedure, this would provide a comparison between instruments, but they don’t. Many researchers are not interested in high readlengths; their work involves smaller sequences, and speed is therefore much more important. The most informative readlength specification includes the sequencing time and a confidence level, usually set at 99%. Therefore, a readlength of 700 means that 700 bases can be identified with 99% confidence as they pass from the capillary during a run. Confidence falls, however, with identification of subsequent bases. The confidence level, or quality of the base identification, can be determined by software. Equipment manufactures supply
their own software, but the best-known package is Phred—a base-calling (baseidentifying) program for DNA sequence traces. It is widely used by sequencing laboratories. For example, Jeff Chapman of Beckman Coulter says that he regularly quotes readlengths with a Phredbased confidence level to customers. Phred’s base-specific quality scoring is considered one of its most innovative features. After calling bases, Phred examines the peaks around each base call and assigns a quality score, with scores ranging from 4 to 60 and higher values corresponding to higher quality. The quality scores are logarithmically linked to error probabilities. For example, Phred 20 is equivalent to 99% confidence.
Detection Fluorescence detection schemes used by the commercial instruments have some fundamental differences. The MegaBACE scans the capillary array with a confocal microscope objective to deliver focused laser light and collect the fluorescent emissions of excited fragments. In this system, the laser scans across the 96 capillaries. The laser beam is focused at the center of each capillary to provide maximum power and ensure that the confocal objective collects emissions from these center positions. Photomultiplier tubes detect the bandpass-filtered fluorescent emissions. The Beckman Coulter instrument has two lasers for excitation (650 and 750 nm) and a photomultiplier tube that detects in the near-IR range. The ABI 3700 uses a sheath-flow detector system and a charge-coupled device (CCD) camera. In this detection scheme, the capillary fits snugly into a tube of optical quartz, which has a square channel slightly larger than the outer diameter of the capillary. A stream of buffer passes through the square channel and over the capillary end, sweeping down analyte and hydrodynamically focusing the stream. The sequenced fragments in the stream are excited and detected as they elute from the anode end of the capillary by laser light focused to a point just below the tip of the capillary. The resulting emissions are dispersed by a reflection grating spectrograph and collected by the CCD camera.
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Spectrumedix also uses a CCD, but the fluorescence is measured on the column, and the detector optics are not scanned. The detector system essentially has 96 stationary detectors in a CCD camera; the CCD arrays are broken down by software into 96 detectors—each monitoring a single capillary. The ABI 3100 also uses this detector design.
The separation matrix The separation matrix functions to resolve DNA fragments in an applied elec-
ment. It also requires a “relaxation” period before sample injection and electrophoresis so that the polymer can regain its native configuration. The injection process also flushes the previous run from the capillaries. Both the Spectrumedix and Beckman Coulter instruments use proprietary LPA matrixes that are loaded by nitrogen pressure. Overall, the time required for LPA matrix injection, relaxation, and sample loading is ~50 min. The POP matrixes are supplied in a bottle that nests inside the instrument
Most of the operating cost is for capillary supply and maintenance. trical field. Predictably, the matrix largely determines the resolution limit for the fragments, but it also dictates instrument design and workflow. The separation matrix is an integral part of the CE sequencer, and a great amount of research has centered on overcoming the undesirable aspects of matrix formulations. Each manufacturer has developed proprietary formulations and preparations optimized for their system. Because the loading, flushing, and lifetimes of capillaries are affected by the matrix, instrument suppliers will not guarantee their equipment unless the specified matrix is used. A linear polyacrylamide (LPA)-based matrix is currently supplied for use with all instruments except the ABI systems. LPA matrix is extremely viscous, but it can routinely obtain readlengths >700 bases. The ABI systems commonly use a substance called performance-optimized polymer 6 (POP-6), but new materials have recently been developed by ABI (cleverly labeled POP-5 and POP-37). These matrixes differ in their polymer sieving ability and, say some experts, the larger pore size of LPA may be more likely to clog with template DNA during electrokinetic injection. In the MegaBACE systems, LPA is packaged in individual tubes and injected into capillaries from a titanium pressure hull under nitrogen gas pressure. This injection requires that a high-pressure nitrogen tank be attached to the instru-
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door of the ABI instruments. Inside the bottle of POP polymer, a supply line injects matrix into the capillaries under applied syringe pressure. No relaxation time follows this low-pressure injection, but subsequent flushing of the cuvette is required to clear excess matrix. The overall processing time between runs for these functions to complete is, again, ~50 min.
Capillaries “The operating cost for these instruments is significant, and most of this cost is for capillary supply and maintenance,” says Paulus. “Labs that do not have a heavy sequencing load are probably better off buying an instrument with fewer than 96 capillaries.” Fused-silica capillaries with coated and uncoated walls are now standard. When LPA is used in capillaries, the capillary walls are often coated because LPA may not wet uncoated fused-silica surfaces, leading to electroosmosis at these surfaces. This extra processing step makes them more expensive, and coated capillaries cost as much as four times that of uncoated ones. The actual costs for capillaries depend on the quantity packaged in a bundle for specific instrument configurations. Replacing defective capillaries and recalibrating the instrument can be a time-consuming process, say users. In the ABI 3700, the capillaries are all part of a single bundle of 104 uncoated, fused-silica capillaries, consisting of 96
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primary capillaries and 8 reserve ones. The reserve capillaries can be designated for use in the event that between one and eight of the primary capillaries exhibit substandard performance. In the MegaBACE, the capillaries are bundled together as sets of 16 coated, fused-silica capillaries; 6 separate bundles comprise the 96. Replacing a bundle requires a careful recalibration. Spectrumedix uses cassettes of 16 uncoated capillaries, which, the company says, can be quickly replaced with little recalibration. Uncoated capillaries last longer than coated capillaries because the coating can degrade with repeated usage. MegaBACE capillaries are specified to provide a lifetime of at least 150 runs, whereas ABI claims that its uncoated capillaries last through 300 runs. In truth, the actual lifetime of capillaries is highly dependent on technique, the matrix, and the types of samples being analyzed.
Versatility is the future According to Chapman, the robust, flexible DNA sequencers will soon be quite common in many smaller laboratories. “Simplify the process, and you reduce the chance of error,” he states. Chapman cites his own company’s eight-capillary sequencer as an example. The system automatically fills the capillary array with LPA gel, loads the sample, applies the voltage program, and calls the sequence. “A host of applications can be run on this instrument, and you don’t have to run 96 capillaries for a simple analysis.” John Fosnacht, marketing manager of Spectrumedix, agrees. “The large genome centers have bought most of their instruments. Now, the direction of research is changing from the large identification labs to smaller research labs that will be buying one machine at a time. These smaller labs will want versatile instruments that perform many kinds of analyses,” he says. Fosnacht provides some examples. “You can screen for mutations 96 at a time, in a very low-cost manner. After you find a mutation, you can do single base extension work, or you can do sequencing,” he says. CE instruments have the ability to do quick quality control evaluations of polymerase chain reaction
product review
replications, DNA sizing and fragment analyses, comparative genotypings, and microsatellite analyses. Forensic applications include identification and, in the United States, CODIS (convicted offender DNA index system) work, which is designed to build a library of offenders. Typical sequencing uses only fourcolor dyes; forensic applications commonly use five. The fifth dye is for an allelic ladder, which serves as an internal standard. Some workers are sequencing with as many as eight colors. They mix two different DNA samples, each reacted with a separate four-dye group. Multiplexing the signals takes advantage of the different sizes of the fragments as well as the various colors, and a single run can analyze two samples at once. “As more and more sequencing data become available, and as the process becomes routine, there will be a move to-
ward personal medicine,” Paulus said. “The differences between two people are expressed by only 0.01% of their DNA. Single nucleotide polymorphism [SNP] analysis will provide the basic genetic signature of an individual. These SNPs can define the differences between people, and they can provide an understanding of individual differences between disease susceptibility and response to treatment.” As CE technology evolves and matures, researchers are also looking ahead to the next generation of sequencers. Paulus thinks that this next generation will also be based on electrophoresis, but that the process will use channels in glass or plastic chips instead of capillaries. “Chip-based systems are integrated subsystems,” he said. “These systems have the promise of getting to high throughputs by faster operation and even higher parallel operation. A typical standard now
is 700 bases per run. On ABI or APBiotech machines, this takes about 3 h. Researchers have shown that on an 18-cmlong chip, you can read the same 700 bases in 20 min. Smaller systems can eventually become portable, and smaller systems require less materials. In my mind, the chip-based device will be a major contender for the next generation of sequencing.” Paulus adds that when sequencing was done with slab-gel systems, costs were about a dollar per base. “This has come down to less than a dime now. I believe the next technology must lower the cost per base down to a penny. This lower cost is now driving the development of chip-based sequencers.” James Smith and Vicki Hinson-Smith are freelance writers based in western Massachusetts.
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