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Applied Biosystems Takes Wraps off Its Microarray Expression Analysis System

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Certain engineers are jokingly known as “belt and suspenders” types — because they wear both on their pants, just in case one system fails.

Applied Biosystems, the Foster City, Calif., DNA sequencing powerhouse, displayed a system of this type as it literally took the wraps off the Expression Analysis System, its entry into the microarray marketplace, last week at the Chips to Hits conference in Boston.

At the root of the beige benchtop system, which uses 2-inch by 3-inch microarrays only readable with ABI instrumentation, is a dual method for detecting gene expression in biological samples — which combines chemiluminescence and fluorescence.

“We decided to look at chemiluminescence because, according to the literature, [it] has tremendous potential for extremely sensitive molecular detection,” Bill Efcavitch, senior director for ABI’s arrays business, said during a presentation on the system on Thursday before a crowd of about 200 people, including scientists and even some competitors.

With a large booth on the main floor of the exhibition hall, and an “executive sponsorship” of what is perhaps the largest trade show for the microarray industry, the company provided the public its first hands-on view of the microarray system it announced in July. The company is also beginning a series of seminars in Europe promoting the system, with sessions in the UK, Sweden, Finland, Denmark, the Netherlands, and Belgium.

And, it provided an insight into the technology it is rolling into an application that enters the estimated $800 million microarray market behind the respective market leaders for pre-spotted arrays — Affymetrix, Agilent Technologies, and Amersham.

The company previously announced that it would first introduce a whole-human-genome microarray by the end of the year. Efcavitch said that rollout is to be followed by the introduction of a whole-mouse-genome microarray in January, and a rat microarray, “later on in the year.”

A One-Color System

The images produced by the Expression Analysis system show light blue dots on a dark background.

“I call it a pseudo two-color system,” Efcavitch said. At the beginning of his presentation, he said of the system: “It is not a scanning system; it is an imaging system. We do a short image, and a long image and then stitch them together to get extended dynamic range.”

Each spot on the gray, gasket-bordered, nylon-substrate-over-glass microarray contains a 60-mer and a 24-mer oligonucleotide, which are manufactured ex situ, and quality-controlled by mass spectrometry analysis before being attached to the nylon substrate.

The 24-mer oligos are the same at each of the [approximately 35,000] spots on the array, he said.

“We use this fluorescence spot at every single spot on the microarray to test for spot finding,” said Efcavitch. “So, even in the absence of a chemiluminescence signal, or the presence at a very low level of chemiluminescence, we are able to precisely locate and define each spot.”

That system of internal control probes allows the system to find each spot and reproducibly map it, he said.

The Expression Analysis System includes a CCD camera and high-powered LEDs for its “pseudo-fluorescent” illumination system, Efcavitch said.

Images are produced in a process that begins with an image of the fluorescence for normalization, then a longer read of the chemiluminescence for the signal.

The fluorescent spots are highly correlated to chemiluminescence spots and act as an internal control for the system — if there is a lack of correlation, the spot is rejected, the company said.

The company on Monday said that it is not prepared to provide further details of its normalization processes.

The Chemistry

According to Efcavitch, the system consists of a model 1700 analyzer, full system software, including an Oracle database, and labeling and detection reagent kits.

The method used in this system for hybridization detection is a biochemical reaction that uses proprietary dioxetane screening reagents from Tropix of Bedford, Mass., a company acquired by PerkinElmer in 1996 and then established as a wholly owned subsidiary of Applied Biosystems.

For labeling, the company said it is offering two sample labeling kits, non-amplification RT labeling (reverse transcriptase) vs. amplifying (reverse transcriptase — in vitro transcription). The processes of generating cDNA or cRNA involve the incorporation of digoxigenin, followed by overnight hybridization to the microarray. After the washing of the cartridge, the process is next followed by the introduction of an anti-digoxigenin, alkaline phosphatase antibody conjugate, a solution that stabilizes and enhances the biochemical light production reaction and produces light, localized to the hybridization, which is then imaged.

“There is no need to really get too synchronized about the addition of the reagents to the readout of the microarray,” said Efcavitch. “The light production will continue for longer than 60 minutes, but then it will slowly use up the substrate on top of the microarray and it will decay off.”

The RT process requires 40 micrograms of total RNA, said Efcavitch, “but we are quite comfortable with 10 micrograms of RNA,” he added.

The RT IVT (amplifying) process requires 1 microgram of total RNA, but “we are quite comfortable with 100 nanograms of RNA,” the company said Monday.

The Data

The ABI expression system can output data into Spotfire, GeneSpring, or flatfile output formats, said Efcavitch.

The product, he added, is intended to be a gene-based microarray, rather than an EST-based microarray. The probes were selected from both the Celera Discovery System database, supplied by Applied Biosystems’ sister company Celera, and from the public sources.

“The Celera process is to computationally predict all 31,097 genes, and then overlay all expressed evidence from public and private sources on top of that, so that we know which transcripts are being detected by each spot,” Efcavitch said.

He gave an example of a particular gene, which he called the protein-5 gene, for which the company defined six transcripts and created a single probe to that gene, to capture all six transcripts, he said.

“So, by having that absolute knowledge of transcripts laid on top of a well-characterized gene, it gives us the confidence to say that we know what it is that we are probing, so that if we get a positive signal, we will be able to map it back to one of these transcripts.”

That information, he said, is provided with the system.

The system uses one probe per gene and “we are absolutely confident that we are probing 60,808 transcripts,” Efcavitch said.

The database included with the system contains a list of every transcript targeted by each probe and a list of public and private identifiers, Efcavitch said.

“We estimate that 95 percent of the transcripts we are looking at have a defined 3’ UTR, so we are fairly confident that we are targeting the 3’ end of the gene,” he said. “We tried to place our probes extremely close to the 3’ end — [they] are, on the average, 432 base pairs from the end of our transcripts.”

The Market

Sources within Applied Biosystems said that the company has sold approximately a dozen of the Expression Array Systems to academic customers. The system is in the CTS phase of marketing - confirmation, test, and setup. The next step for the product is official NPR (new product release), which depends on filing of ISO (International Organization for Standardization) and CE documentation.

Others say that the product will need automation in order to penetrate the pharmaceutical marketplace, while academic customers will require a robust system of customer service and support.

The instrument is list priced at $179,000, and the arrays are expected to sell at industry average pricing ($500 per array). The appearance at Chips to Hits was regarded as a product preview, not a product launch. The ABI website contains no information to document the Chips to Hits presentation, but does include a page with information on the system.

— MOK

 

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