Readers In The Storm: A Profile Of Automated DNA Sequencers

Date: November 10, 1997 Chart 1 Chart 2 It was a busy evening at The Eagle. Less than half a century ago, inside this small Cambridge pub, two gentlemen raised their glasses to salute their remarkable genetic findings. At the time, the two men (and a few close colleagues) were the only souls on the planet privy to the molecular structure of DNA. Does it seem odd, then, that researchers around the globe could be toasting the description of the entire human genome less than ten years from now? You would be hard pressed to find a more high-tech, precision instrument in a biotech lab than an automated DNA sequencer. Using precision lasers and sensitive detection devices, these instruments are more finesse than brute force. DNA sequencers are likely to be set aside from all other pieces of laboratory hardware, having benchtops or entire rooms devoted to their presence. Similar to cracking the binary code of a computer, these tools of technology are burdened with the task of determining patterns of the four bases of DNA. Following electrophoretic separation, a laser excites fluorescent-labeled DNA fragments. Detectors then capture the fluorescence emission, and, depending on the signal generated, the fluoresced signal corresponds to one of the four bases. Some sequencers incorporate "four dye-one lane" technology, allowing four different labels to be used in each lane or capillary. As biotechnology screams into the 21st century, the term "high throughput" is becoming more commonplace in a variety of applications. Nowhere is this more apparent than in the field of automated sequencing, thanks in part to the race to sequence genomes. The task of sequencing multiple genomes has prompted manufacturers of sequencers to optimize performance in their instrumentation and has also spawned some unique and specialized sequencing tools around the world. Researchers with sequencing needs can still rely on several DNA sequencing services, but automated DNA sequencers have quickly become a standard piece of laboratory equipment. The genomyxLRSTM From Genomyx Air Apparent Beckman/Genomyx genomyxLRTM High Resolution DNA Sequencing System genomyxLRSTM Fluorescent Imaging System The genomyxLR DNA Sequencing System is the flagship product of Genomyx Corporation (Foster City, Calif.), a wholly owned subsidiary of Beckman Instruments. The genomyxLR provides long-read, high-resolution isotopic DNA sequencing and differential display. According to Genomyx, the resolving power and long-read capability of this system stem from a proprietary method of maintaining uniform temperature throughout the 61 x 33 cm gel during electrophoresis. The "air impingement" method of temperature control employs a patterned array of air delivery jets that allows optimum control of gel temperature, eliminating band diffusion, smiling, and other distortions. This method permits voltage and temperature to operate independently of one another during electorophoresis, allowing long, low-voltage runs, which increase resolving power. The genomyxLR features integral microprocessor control, internal gel drying, and a 3,500-volt power supply with safety interlocks. The Isotope Capture Cartridge (ICCTM) automatically isolates liquid radioactive waste for subsequent disposal. The genomyxLRS integrates the genomyxLR DNA Sequencer and the genomyxSC Fluorescent Imaging System for high-resolution DNA fragment analysis. The genomyxSCTM Fluorescent Imaging Scanner is designed to provide a fluorescent detection and analysis system that can be used for a wide variety of molecular biology techniques. The package supports applications such as STR/genotyping, AFLP, RFLP, and mutation detection. The genomyxSC is controlled by acquireSC software, which collects data and converts it into an 8-bit TIFF file. Data can then be imported into one of two Genomyx software products-extendLRS for DNA base calling, or claritySC for molecular weight determination and fragment analysis. The genomyxSC can also scan and digitize autorad films, allowing digital image analysis of data generated from isotopic or nonisotopic methods. HUGE Success GeneSys Technologies GTI-9600TM The GTI-9600TM from GeneSys Technologies (Sauk City, Wis.) employs Horizontal Ultrathin Gel Electrophoresis (HUGE) technology to enhance performance. HUGE utilizes ultrathin 75-micron gels, allowing higher voltages during electrophoresis. The HUGE system also employs a water-cooled cassette, alleviating problems such as smiling and frowning associated with temperature gradients across the gel. By connecting the cassette to the water circulator during polymerization, gels can be cast under controlled temperatures. The instrument uses a 100 mW argon ion laser connected to an optical fiber that delivers the excitation beam to an optics assembly on the scanning stage. The laser beam is subsequently focused to a 100-micron spot on the horizontal gel at an angle optimized to reduce reflection (Brewster's angle). Fluorescence emitted from labeled DNA fragments is collected using a 40X high numerical aperture microscope objective, mounted on the scanning stage, and focused onto another optical fiber, which directs the fluorescence to a detector array. Composed of appropriate light filters, dichroic mirrors, and photomultiplier tubes, the patented simultaneous four-color detector collects 1,000 pixels across a gel every 0.75 seconds, facilitating accurate lane finding and identification of fragments. The GTI-9600 can separate and resolve single-stranded DNA molecules between 25 and 620 bases in length from each of 96 lanes in less than 90 minutes of electrophoresis. GeneSys offers CartographerTM software for genotyping and fragment analysis. Cartographer performs automatic lane tracking, signal extraction, color deconvolution, molecular weight determination, and DNA fragment sizing. Raw data is presented through a graphical user interface and stored in an AccessTM database. Users view results in real time and can always access raw data. GeneSys is also developing software packages for gene expression and base calling of DNA sequence data. GeneSys has recently developed ReLoad, a reusable buffer system that allows up to five electrophoresis runs using the same HUGE gel. Lastly, GeneSys Technologies offers customized robotics for laboratories with automation needs. The GeneLab, available this year, consists of a robotic arm to move DNA samples from one location to another to fully automate the chemical preparation of DNA prior to analysis on the GTI- 9600. The GeneLab consists of modules, such as pipetting stations, thermal cyclers, and centrifuges, that are specified by customers for their particular applications. Sequencing North and South LI-COR Biotech IR2 Long ReadIR System (4200L) IR2 Gene ReadIR System (4200S) LI-COR systems combine infrared fluorescence chemistry with laser technology and software to produce high-performance systems for DNA sequencing and genetic analysis. LI-COR (Lincoln, Neb.) claims infrared fluorescence detection optimizes sensitivity since most gels and soda lime gel plates exhibit low fluorescence at infrared wavelengths. Concurrently, low background fluorescence at the detection wavelength provides a dynamic detection range that enhances the ability to resolve weak and strong bands on the same image. LI-COR sequencers utilize a scanning laser microscope assembly which uses diode lasers, similar to those used in compact disk players. A highly sensitive, thermally controlled, solid-state avalanche photodiode detects the fluorescence generated from excited, dye-labeled DNA fragments in the gel. Unlike systems with a fixed focal point, the laser and fluorescence microscope are mounted on a focusing stage that is automatically focused on the center of the gel at the beginning of each run. This means the microscope can be refocused at the point of optimal detection when gel thickness is changed or some other variable warrants repositioning the scope. IR2 systems allow Simultaneous, Bi-Directional Sequencing (SBSTM), enabling users to combine forward and reverse primers in one reaction to sequence both directions on the same template. Potentially, this can save users at least 50% on reagents, gels, and labor. Inserts of up to 2 kb can be sequenced in one SBS reaction. LI-COR offers both single- and dual- laser systems. The dual- laser system uses two lasers and detectors for independent infrared fluorescence detection from each dye used. According to the company, IR2 images exhibit high fidelity due to elimination of signal crossover between channels. Fluorescence from each dye is separated by over 110 nm, and each detector is filtered to detect fluorescence from only one of the dyes. Gene ImagIRTM software allows fragment analysis and provides a relational database. A standard pedigree table lets researchers track family relationships, affection status, liability classes, and more. Templates allow lanes to be located in seconds, and each sample is automatically identified based on the template. Alleles are also quickly identified and sized using molecular weight standards; bands can also be quantified using concentration standards. After analysis, Gene ImagIR can generate linkage or mapping files. Power to the Polymer PE Applied Biosystems ABI PRISMTM 310 Genetic Analyzer ABI PRISMTM 377 DNA Sequencer ABI PRISMTM 373XL Upgrade The ABI PRISM( 310 Genetic Analyzer from PE Applied Biosystems (Foster City, Calif.) automates all aspects of DNA sizing, quantitation, and sequencing. The ABI PRISM 310 uses a flowable polymer-as opposed to acrylamide or agarose-which is injected into the capillary before each run. The system automatically injects samples in the specified order, runs the separation, and analyzes the data. A line of ready-to-use Performance Optimized Polymers is available for DNA Sequencing (POP-6) and high-resolution denaturing DNA fragment analysis applications (POP-4). The capillary lasts for at least one hundred sample runs (and in many acases, hundreds of runs) and can be replaced in minutes. An argon ion laser excites the multicolor fluorescent dyes, which are detected by a CCD (charge-coupled device) Camera Detector. Preprogrammed software modules were developed in order to streamline system control and data management. All functions are controlled from the system computer using point-and-click commands and pop-up menus. The system automatically controls run temperature, time, and voltage. Depending on the application and required resolution, results for the first sample are available in as little as 12 minutes from sample injection. The ABI PRISM 377 DNA Sequencer automatically analyzes DNA molecules labeled with any of four fluorescent dyes. After samples are loaded onto the system's vertical gel, they undergo electrophoresis, laser detection, and computer analysis. Separations can be viewed on-screen in real time, and final data can be printed in a variety of formats. The 377 accepts 12, 36, and 48 cm gels so run times and sample resolution can be optimized according to analysis type, either DNA sequencing, molecular sizing in base pairs, or molecular quantitation. The 377 consists of an electrophoresis and detection unit, a Power Macintosh computer, an optional color printer, and software for DNA sequencing analysis or PCR fragment sizing and quantitation (GeneScan¨). Although the ABI PRISM 373 was replaced with the 377, PE Applied Biosystems provides an upgrade package for the ABI PRISM 373. The upgrade re-engineers a 373 to collect twice as many channels-from 194 to 388-with each laser scan. This allows analysis of up to 48 or 64 samples of fragment or sequence data. The upgrade includes a more powerful Power Macintosh computer and a Translation Interface Processor that buffers the memory from the 373. Also included are more intuitive data collection software, the latest version of DNA Sequencing Analysis and optional GeneScan Analysis application software, status lights for quick instrumentation checks, and necessary accessories. DNA Sequencing Analysis software features a new user interface to simplify operation, stop point algorithms that let the user define where base calling stops, base calling algorithms for a variety of sample types and run conditions, and automatic lane tracking. ABI Sequencers carry UL, CSA, and CE approvals, and PE Applied Biosystems provides world-wide service and technical support. Pharmacia Biotech's ALFexpress Combining Sci-Fi with Cy5 Pharmacia Biotech ALFexpressTM DNA Analysis System The ALFexpress DNA Analysis System from Pharmacia Biotech (Piscataway, N.J.) is a fully automated electrophoresis system with a sophisticated detection system based on laser- induced fluorescence. The fluorophore used for detection in the ALFexpress System is the fluorescent carbocyanine dye Cy5(. This dye possesses a high quantum yield, which results in high signal-to-noise ratios. Also, the single-dye format yields easily interpretable raw data, permits accurate and easy detection of heterozygote mutations, and eliminates mobility differences between fragments due to different labels. ALFexpress utilizes a red helium neon laser (633 nm) that penetrates the entire width of the gel. The laser beam automatically adjusts its position to maximize beam intensity through the gel. Forty fixed photodiodes-one for each lane located at right angles to the laser beam-measure the fluorescent emission from the labeled DNA fragments as they pass by the detector. In many cases, ALFexpress can read 300 base pairs with 99% accuracy in approximately 1 hour. The ergonomic design optimizes physical access, and a tilt-up cover minimizes bench space needed to access samples. ALFexpress is controlled via a PC, running under a multitasking operating system. The ALFexpress Cooler Kit allows the connection of the external thermostatic circulator MultiTemp III for single- strand conformation polymorphism (SSCP), dideoxy fingerprinting (ddF), and heteroduplex studies. After installation of the Cooler Kit, the ALFexpress system can operate between 4° and 60°C. ALFwinTM software controls the instrument and evaluates sequencing data, while Fragment Manager evaluates fragment data. ALFwin gives users total and simultaneous control over two ALFexpress and ALF series automated sequencers. Data can be evaluated, processed, and exported for use with other software applications. AlleleLinksTM is a research tool for linkage analysis, bacterial and viral typing, forensics and paternity testing, animal and plant breeding, mutation analysis, and more. This program is compatible with such research techniques as STR/VNTR, RAPD, DDRT, and SSCP/ddF. Mutation Analyser is for use with mutation studies seeking specific base aberrations in sequenced RNA or DNA. Lastly, HLA SequiTyperTM is suited for transplantation medicine, analysis of disease susceptibility, paternity testing, and anthropological studies. Cy5 is a trademark of Biological Detection Systems, Inc. This dye has the same properties as Dye667. The Open-Gene Automated DNA Sequencing System from Visible Genetics Blasting Genes Wide Open Visible Genetics, Inc. OpenGene Automated DNA Sequencing System The OpenGene System from Visible Genetics (Toronto) is focused exclusively on the unique needs of clinical laboratories. The 16-lane ultrathin (14 x 14 cm) MicroCel cassette is central to the OpenGene system's speed and efficiency. A 50-micron acrylamide gel is cast between the MicroCel's two glass plates to facilitate rapid DNA electrophoresis. Acrylamide is supplied premixed in SureFillTM disposable cartridges. The MicroCel is also disposable, eliminating disassembly and plate cleaning. The Gel Toaster photopolymerizes the MicroCel in 3 minutes. At the heart of the OpenGene system is the MicroGene Blaster Automated DNA Sequencer, a compact, fluorescence-based DNA sequencer designed for rapid electrophoresis. With fixed photodiodes and no moving parts in the detection system, the sequencer has a high signal-to-noise ratio and requires minimal servicing. The OpenGene system uses single dye primer CyTM5.5 chemistry, which possesses a high signal-to-noise ratio. Visible Genetics recommends Amersham's Core Sequencing Kit for general sequencing needs or their own GeneKits for specific analysis needs to ensure accurate and reproducible sequencing results with consistently even peaks. Including the 5 minute procedure of MicroCel cassette preparation, the OpenGene system has a total time-to-result of just over 35 minutes, allowing sequencing of 300 bases. GeneObjects DNA analysis software provides a comprehensive system for DNA sequencing and fragment analysis tailored specifically for clinical labs. GeneObjects not only manages electrophoresis but also analyzes sample data and organizes customer-defined databases for comparative analyses and generation of output reports. Intel-based UNIX workstations allow high analysis throughput and multitasking functionality. GeneObjects can manage multiple MicroGene Blaster sequencers. The Gene LibrarianTM tool allows comparison of a sequenced allele to a whole database of known variations and can identify the best match. The ComparatorTM tool allows a test assay to be compared with a standard and highlight areas of difference. Some specially developed sequencing machines have been constructed during the past few years. A high-speed DNA sequencer, the ESY9600 Multiplexed Capillary Electrophoresis DNA Sequencer, developed by the U.S. Department of Energy's Ames Laboratory at Iowa State University, has been licensed to SpectruMedix (formerly PATCO; H. Surface, Laser Focus World, July 15, 1995, page 3) in State College, Pa. The multiplexed fluorescence detector for capillary electrophoresis was developed by Edward Yeung and uses cartridges of 96 fused-silica capillaries for electrophoresis and a PC with software for automation control and data processing. The sequencer simultaneously performs excitation and detection; fragments are detected using an argon laser and a CCD camera. According to a September 1997 interview with Senior Scientist Dr. Thomas Kane, "SpectruMedix will be releasing prototypes to selected sites at the end of the year, and systems could become commercially available early next year." An automated, adaptable system for high-throughput genomics was developed at the Whitehead Institute (Cambridge, Mass.) and dubbed the Sequatron (T.L. Hawkins et al., Science, 276:1887-1889, 1997). Currently, three Sequatron systems perform (1) DNA purification from M13 phage and PCR products, (2) DNA sequencing reactions using dye-primer and dye- terminator chemistries, and (3) custom PCR amplifications and sequencing reactions on selected templates to close gaps in partially completed genomic sequences. The purification Sequatron has a throughput of 16,000 templates per day with unattended operation. The sequencing Sequatron has similarly sequenced more than 16,000 samples in one day. GeneTrace Systems (Menlo Park, Calif.) has developed a system based on laser-based time-of-flight (TOF) mass spectrometry for DNA sequencing and sizing (J.A. Monforte et al., Nature Medicine, 3:360-362, 1997). Although the instrumentation is not commercially available, this technology is offered by the company as a service for DNA sequencing. Utilizing this method, DNA is mixed with a light-absorbing matrix molecule and placed in a vacuum chamber in a mass spectrometer. A nanosecond-long pulse of ultraviolet laser light irradiates the sample, causing a microscopic puff of matrix molecules to desorb off the plate, and, in an electric field, the charged DNA molecules are accelerated toward the detector, with lighter molecules having a shorter time-of-flight. Mass is proportional to the square of transit time. The author can be reached online at sbeck@the-scientist.com. Chart 1 Chart 2

Readers In The Storm: A Profile Of Automated DNA Sequencers

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