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ACTATCGAA
Knowing the Code Manual and Automated Gel-Based DNA Sequencers Whether one is using a manual or an automated sequencer, electrophoretic DNA sequencing starts with the same polymerization and sequencing reactions that result in samples that have been labeled (either radioactively or by fluorescence) according to the base sequence. The process of casting the gel, loading the templates on the gel, and performing the separation is also nearly identical. Slab-gel DNA sequencers use a polyacrylamide gel that is injected between two glass plates. The gel is allowed to polymerize around combs, which create wells or lanes. Once the gel has polymerized, the DNA fragments from the sequencing reactions are pipetted into the wells. For all manual and most automatic sequencers, a single sample requires the use of four lanes—one for each of the bases found in DNA A voltage is applied, and the DNA fragments are separated by electrophoretic mobility. Given that so many procedures are shared by manual and automated sequencers, one might ask, "Just where do automated sequencers get their throughput advantage?" The answer lies in the
Deciding whether to purchase an automated or a manual sequencer hinges largely on throughput steps that follow the electrophoretic separation. Manual sequencers require that the gel be dried (a process that requires at least 15 min) and exposed to autoradiographic film for at least 18-48 h, and sometimes for as long as two weeks. At that point, the film must still be analyzed. In contrast, automated sequencers identify the base sequence as the fragments migrate past the detector and provide "real-time" computerized base-calling. The time saved by automated DNA sequencers is considerable. For example, a fragment that might require 4 days to
sequence manually can be done in only 4 hours with an automated sequencer. Analytical Chemistry asked John C. Ford, an assistant professor of chemistry at Indiana University of Pennsylvania; Peter Oefner of the DNA Sequence and Technology Center at Stanford University; Barry L Karger, professor of chemistry at Northeastern University; and Christopher R. Somerville of the Department of Plant Biology at the Carnegie Institution of Washington (Stanford, CA) for their views on manual and automated DNA sequencers. Tables 1 and 2, although not necessarily comprehensive, list important features of representative manual and automated DNA sequencers, respectively. Table 2 includes Perkin Elmer Applied Biosystems' ABI Prism 310, which, unlike the other instruments, is an automated capillary sequencer that uses flowable, reusable polymer matrices rather than cast gels. Practical considerations Purchasers of manual sequencers are more concerned than users of automated sequencers about gel casting, gel size,
Analytical Chemistry News & Features, August 1, 1996 4 9 3 A
Product
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Table 1. Summary of representative manual DNA sequencers Model
Sequi-Gen GT
Company
Bio-Rad 2000 Alfred Nobel Dr. Hercules, CA 94547 510-741-1000
Price $850-$970 Dimensions instrument ( H x W x D ) Varies with cassette Cassettes 21 x 40, 21 x 50, 38 x 30, 38 x 50 cm Buffer volume Upper (Cathode) 500 mL-1400 mL (minimum) Lower (Anode) 350 mL (minimum) Combs 24-, 34-, 36-, 48-, 49-, 68-, 73-, 97-, and 100well sharkstooth; 16-, 20-, 32-, 36-, 44-, 60-, and 80-well conventional Spacers 0.25, 0.4, 0.75 mm standard and 0.25-0.75 mm wedge Temperature control Upper buffer chamber covers entire gel area, acting as a heat sink Detection method Autoradiography Special features
Horizontal gel-casting method; IEC1010 safety certification; buffer-backed design ensures uniform heat distribution
Reader service number 401
and ease of buffer disposal, although the characteristics will affect both groups of users. Because gel casting is the main source of failure in manual sequencing, ease of casting the gel is very important for researchers who use manual sequencers because it reduces costs in terms of both time and reagents. Ford says, "In the typical [high-volume] laboratory, you have one or two technicians who are casting the gels all the time." But in smaller laboratories such as Ford's, the regular turnover of graduate and undergraduate students means continually teaching new people to cast gels, resulting in a much higher frequency of gel failure. Oefner who uses automated sequencers exclusively says "All of us want to get rid of 494 A
Base Runner 100 and 200 Eastman Kodak P.O. Box 9558 New Haven, CT 06535 203-786-5600 www.kodak.com
BioMax STS-45i
genomyxLR
Hoefer SQ3 Sequencei
Eastman Kodak P.O. Box 9558 New Haven, CT 06535 203-786-5600 www.kodak.com
Genomyx 353 Hatch Dr. Foster City, CA 94404 415-572-8800
Hoefer Pharmacia Biotech 654 Minnesota St. San Francisco, CA 94107 415-282-2307 www.biotech. pharmacia.se $840
$895 (100); $1495 (200) $795
~ $9,000
75 x 30 x 30 cm Adjustable
4 8 x 4 9 x 2 2 cm 45 x 37.5 cm (outer); 43 x 37.5 cm (inner)
7 9 x 8 1 . 5 x 5 1 cm 61 x 33 cm
4 8 x 4 1 x 2 2 cm 41.9 x 33.3 cm (outer); 39.4 x 33.3 cm (inner)
500 mL
530 mL (maximum)
125 mL
400 mL (minimum)
500 mL 32- and 48-well sharkstooth; 32-well conventional
530 mL (maximum) 64- and 96-well sharkstooth; 32-and 64-well conventional
250 mL 48-, 64-, 70-, and 96well sharkstooth; 50and 63-well flat well
350 mL (minimum) 32-, 64-, and 96-well sharkstooth; 32-well standard
0.2 and 0.4 mm standard; 0.2-0.8 mm and 0.4-0.8 mm wedge Thermocore plate
0.2 and 0.4 mm standard; 0.2-0.8 mm wedge White aluminum thermoplate
0.2 and 0.34 mm standard; 0.2-0.4 mm wedge Air impingement
0.2, 0.4, and 1.0 mm standard; 0.2-0.5 mm and 0.2-0.75 mm wedge Aluminum plate
Autoradiography
Autoradiography
Adjustable heights; slant-back design that holds glass plates in place during assembly; two-sided (Base Runner 200) with gels on both sides
Slant-back design so that glass plates stay in place during assembly; white thermoplate eases visual inspection of run progress
Autoradiography and Autoradiography two-color fluorescence Optional genomyxSC Certified safety interlock fluorescence scanner that digitizes data from gel and allows nonradioactive measurement in a manual sequencer; isotope
403
capture cartridge that adsorbs excess radiolabel from lower buffer chamber 404
402
cast gels. That is why everybody is getting excited about reusable matrices [such as those offered by the ABI310]." The question of gel size affects users of manual and automated sequencers because the length determines the maximum length of the DNA sequence that can be resolved and the width determines the number of samples that can be analyzed in a single run. Users of manual sequencers, however, also need to be aware of the size of the autoradiographic film. Some systems have adjustable gel sizes with a single cassette, and other systems offer interchangeable cassettes. Ford does not believe, however, that adjustability really provides an advantage. He also recommends (unless one is cer-
Analytical Chemistry News & Features, August 1, 1996
405
tain that the applications will all require shorter gels) purchasing the largest size that can be used with standard autoradiographic film. "You don't save significantly by using a smaller size," Ford says. "If [the gel size] goes beyond the standard size film, it's a big disadvantage." Annette Tumolo, product manager of Bio-Rad's Genetic Systems Division, says that they make some of their gel cassettes slightly longer than the standard film (50 cm for the gel rather than 40 cm for the film), because the top 10 cm of 3. gel tends to contain unresolved bases. A third factor that may influence the decision about manual sequencers is the ease of buffer disposal. This is particularly important because manual sequencers
Table 2. Summary of representative automated DNA sequencers Model Company
Price
Models 4000L and 4000S LI-COR Biotechnology Division 4308 Progressive Ave. Lincoln, NE 68504 402-467-0700 www.licor.com $69,500 (4000L) $49,500 (4000S)
Dimensions Instrument (H x W x D) 92 x 53 x 41 cm (4000L) 59 x 48 x 41 cm (4000S) Length to detector 8, 15, 23, and 31 cm (S and L); 56 cm (L) Lanes 64 Buffer capacity Upper (Cathode) 500 mL 500 mL Lower (Anode) Temperature control Aluminum thermoplate; 40-60 °C Labeling strategies IR-labeled primer or IRlabeled dATP
ABI PRISM 377 DNA Sequencer Perkin Elmer Applied Biosystems Division 850 Lincoln Centre Dr. Foster City, CA 94404 415-570-6667 www.perkin-elmer.com INA
ABI PRISM 310 Genetic Analyzer Perkin Elmer Applied Biosystems Division 850 Lincoln Centre Dr. Foster City, CA 94404 415-570-6667 www.perkin-elmer.com INA
ALFexpress DNA Sequencer Pharmacia Biotech 800 Centennial Ave. Piscataway, NJ 08854 908-457-2222 www.biotech.pharmacia.se
81 x 74 (door closed) x 52 cm
86.36 x 60.96 x 55.88 cm
80 x 49 x 53.5 cm
12, 36, and 48 cm
50 cm
15 and 30 cm
_36
Single capillary
600 mL 700 mL Thermoplate; ambient -60 °C
4 mL 9 mL Thermoplate; ambient -60 °C
INA
__4? 1000 mL 1000 mL Thermoplate filled with water; from ambient + 10 °C to 60 °C Cy5-labeled dATP
Four colors of 5'dye-labeled primers or dye-labeled terminators IR fluorescence with solidVisible fluorescence with argon state IR laser diode (60,000- ion laser and CCD detector h lifetime) and avalanche photodiode detector (Si)
Four colors of 5'dye-labeled primers or dye-labeled terminators Visible fluorescence with argon ion laser and CCD detector
3-4 h (25-cm gel) 450-600 48 (3 gels) (8-h day)
2.25-3 h (36-cm gel) 450-600 108 (3 gels) (8-h day)
2.5 h (50-cm capillary) 600
6h 600-750 40 (4 gels) (24-h day)
10-16 h (56-cm gel) 800-1200 32 (2 gels) (8-h day) Sequencing, genotyping, mapping, mutation screening, forensics, HLA analysis
7.8-9 h (48-cm gel) 700-600 72 (2 gels) (8-h day) Sequencing, genotyping, mutation screening, linkage analysis, loss of heterozygosity
12 h 800-1200 20 (2 gels) (24-h day) Sequencing, genotyping, mutation screening, quantitation
Pentium computer with OS/2 Warp; Base ImaglR sequencing software; Gene ImaglR genotyping and fragment-analysis software; data in both autorad image and curve formats; realtime base calling; ambiguous base calls automatically marked during sequencing; 99% base-calling accuracy Special features Up to three sequencers controlled by one PC; automatic focusing for gels of different thickness; no gel or laser alignments; soda lime glass plates Reader service number 406
Apple Power Macintosh included with system; gel image displayed during data collection; software transfers data automatically to DNA Sequencing Analysis software; base-calling algorithms for single- and double-stranded DNA, PCR fragments, and long read lengths; 98.5% base-calling accuracy Four-dye, one-lane sequencing; application-specific software
NA NA NA Sequencing, genotyping, mutation screening, DNA fingerprinting, single-strand conformation polymorphism, quantitation Apple Power Macintosh included with system; systern automatically controls analysis conditions; data automatically transferred to analysis software; continues to run samples while data from previous runs are analyzed
Four-dye, one-lane sequencing; application-specific software; no gel pouring; automated sample loading
Application-specific software; self-aligning gel cassette
408
409
Detection method
Fast sequencing Time Bases/sample Samples loaded/day Long sequencing Time Bases/sample Samples loaded/day Applications
Software
407
Visible fluorescence with helium-neon gas laser and 40diode GaAsP array; 15,000-h laser lifetime
OS/2 multitasking environment that requires 486 processor, 66 MHz, 16 MB RAM (included with system); produces raw data that can be analyzed without processing, if necessary; marks ambiguous base calls; 99.5% base-calling accuracy
Analytical Chemistrv News & Features. Auaust 1. 1996 4 9 5 A
Product
Review
normally use radioisotope labeling as the means of detection. Ford says, "Draining the buffer solutions should be as easy as possible to avoid spills. The lower reservoir in particular (because excess radiolabel ends up there) should be capable of being drained while on the apparatus—that is, in place—via a valve, rather than being manually removed." Because radioisotope labeling is hazardous and disposal is expensive, the use of nonradioactive fluorescent labeling is one advantage automated sequencers offer. Genomyx makes a manual sequencer that can also be used with fluorescent dyes and imaged using a scanner. Because the fragments remain in the gel, the separated fragments can be excised for further analysis, unlike automated sequencers, in which the fragments are detected as they migrate off the gel. Temperature control
Temperature uniformity and heat dissipation across the gel are important for both manual and automated sequencers. Temperature variations can create differences in the electrophoretic mobilities between lanes. The outer lanes usually cool more rapidly than do the center lanes, and the fragments then migrate more slowly in the outer lanes. The templates in the center lane can reach the end of the gel as much as 20 bases ahead of the outer lanes, resulting in base patterns known as "smiles." Although "smiling" does not make the gels impossible to read, it does make reading difficult and is undesirable. Thin gels dissipate heat more quickly than do thick gels, but according to Oefner, ultrathin gels are not used regularly because they are harder to cast. The faster the run, the more important heat dissipation becomes. Oefner says, "If you want to run faster, you have to make sure you have sufficient heat dissipation. In the room where we keep our automated sequencers, we discovered that the [air conditioning] wasn't enough. Now we have a vent that goes directly outside." Most manual sequencers control temperature uniformity with an aluminum plate attached to the glass plates housing the gel. Ford says that this is the type of system he uses; he still sees "smiling" in his sequencing runs. Another way of achieving temperature uniformity for the manual sequencers is through the use of an upper buffer reservoir that is in contact with the entire area of the gel plate. Temperature regulation has also been achieved through the use of high-velocity, turbulent airflow across 496 A
the gel from an array of air jets that are controlled by a separate heating element and are thus independent of the applied voltage—a technology that is known as "air impingement." A question of throughput
The decision to sequence manually or automatically hinges ultimately on laboratory throughput. Ford says, "For a lab that can afford it, automated is the way to go as long as the throughput can justify it." He notes, however, that manual sequencers have an advantage in terms of price; they require a "much lower capital outlay," he says. "You pay for it in terms of [greater difficulty in] reading the sequence, but there are ways to get around that with scanning." Scanners digitize the data from either the autoradiographic film or from the gel itself for subsequent computer manipulation. Ford continues, "It is my impression that people [running re-
Running three or more gels in a week can justify automation. search programs] at the low [throughput] end, like those at my lab, would never dream of buying an automated sequencer. Certainly, for $5000, you can start manual sequencing." Oefner quantifies the number of gels that would justify the purchase of an automated sequencer: "If you want to run three or more gels every week, get an automated system." However, he also cautions that "if you have an automated sequencer, you have to run it," because the overall sequencing performance deteriorates if the system remains idle. "For most small labs," Oefner says, "automatic sequencers are overkill. They just don't need that sort of throughput." Karger says that the better performance can be attributed to a number of factors, not all of which are instrument related. He says, "You develop a betteroiled protocol if you are using [the instrument] all the time." Karger notes that automated sequencers are often found in a core facility or are shared by several indi-
Analytical Chemistry News & Features, August 1, 1996
vidual researchers rather than being located in an individual laboratory. The advantageous increase in throughput allowed by automated sequencers naturally increases the number of samples that must be loaded, creating a new, if possibly minor, problem. Oefner says, "Manufacturers of automated slab-gel DNA sequencers have to pay more attention to the loading of gels, especially with the increasing number of lanes that can be run per gel. To load manually up to 1801-uL samples per sequencer per day for six days a week becomes very strenuous for [a technician's] hands and eyes." Automated capillary DNA sequencers provide a bridge between manual sequencers and the high-throughput automated slab-gel DNA sequencers. The capillary instruments do not require pouring a gel and thus eliminate one of the laborintensive tasks of slab-gel sequencing. In addition, capillary sequencers automatically inject the sample into the polymer matrix so that the hand and eye strain to which Oefner refers is reduced. Somerville says that researchers who want to run 10-12 sequences every day should consider a capillary sequencer. "Capillary sequencing brings automated sequencing to the individual lab. We have 20 students and post-docs who all know how to use it," he says. "If you're making a construct and just want to check the junction, you can quickly sequence the sample." Somerville says that capillary sequencers have a lower throughput than manual slabgel sequencers. Capillary sequencers allow longer read lengths than most manual sequencers and shorter read lengths than automated slab-gel sequencers. By using visible or IR fluorescent dyes, automated sequencers (both slab-gel and capillary) detect the DNA fragments in real time as they migrate past the detector. Fluorescent dyes have been used in four-dye, single-lane analysis and singledye, four-lane analysis. The primary advantage of a four-dye analysis is the fact that four times as many samples can be loaded on a single gel, thus increasing throughput. The four-dye technology also makes possible the single-capillary sequencer. Karger indicates, however, that single-dye analysis, which does not allow as many samples as four-dye analysis, often allows longer read lengths. IR dyes also provide higher S/N and allow the use of solid-state lasers and detectors, but have not been used in single-lane analysis. Although four-dye, single-lane analysis would seem to complicate the sequencing
algorithms and introduce more possibilities for ambiguous base-calling, Oefner says, 'Today the software and sequencing chemistry are so much better [than they used to be] that [ambiguity] is never really a problem." According to Karger, "The software's very good now at correcting for mobility shifts. New developments will be available from the Human Genome Project, where there's a continual effort to try to improve the software." Looking ahead
DNA sequencers are not just for sequencing anymore. Sequencers can be used for other techniques, such as differential display, genotyping (microsatellite analysis), and mutation screening. As the map of the human genome comes to fruition, these forms of DNA analysis will take priority over sequencing. One or two microliters are usually loaded on a gel-based sequencer, but larger volumes are used in the reaction. "You waste a lot of money running reaction volumes that you don't need," Oefner says. "This will be even more true for capillary sequencers unless the sequencing reactions are accordingly scaled down. That's the only way we can really reduce the cost of [DNA sequencing]." Oefner sees the future moving to faster, more cost-efficient methods because of the expense of materials and the time required for DNA sequencing. "[For genetic screening], we want to know the differences [between sequences], not the bases that are the same. People will be looking for very cheap, automatic ways to compare two sequences." He believes that methods such as HPLC and enzyme mismatch cleavage will be used to screen quickly two sequences to determine whether they are identical which is especially important with disease-causing mutations. Karger and points out that other methods including electrophoresis will also be used to rapidly screen for mutations particularly larger percentap/e of the genome sequence is known Only after genes have been determined to differ will they be sequenced Karger sees the future of DNA sequencers in capillaries and microchannel plates. "Capillaries or, alternatively, grooves on a flat plate that mimic capillaries will play a role, allowing higher fields and faster separations," he says. He also sees chemical arrays for sequencing ("sequencing on a chip") playing a role, especially in cases of known sequences. Celia Henry
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