By David N. Leff
How much genomic information can fit on the head of a pin?
This question, unlike the one about dancing angels, is not theological, but biological.
Scientists at Sequenom Inc., of San Diego, Calif., dipped the 400-micron head of a metal pin into a microtiter plate well to make surface contact with a fragment of the p53 gene sequence. The pin transferred that nano-scale dab of nucleic acid to a DNA-array chip for testing by mass spectrometry.
"This piece of p53," observed biochemist Maryanne O'Donnell, group leader of chip development at Sequenom, "was just the first example of the company's DNA MassArray technology — actually, a demonstrator model."
She is a co-author of a report on this exercise in the April 1998 issue of Nature Biotechnology. Its title: "Sequencing exons 5 to 8 of the p53 gene by MALDI-TOF mass spectrometry."
O'Donnell explained that she and her co-authors chose p53 to showcase their proprietary procedure "because it's a small, well-studied model system with lots of mutations. Incidentally, those mutations relate to colon cancers. If the mutation of a gene that you're looking for is known, then what you do with our MassArray application is sequence that portion of the gene to see if it's mutated."
Since p53 won its oncogenic Oscar back in 1994, more than 6,000 mutations in its gene have been reported. "We selected exons 5 through 8 to analyze," O'Donnell said, "because this is where most of the cancer-related mutations cluster."
Sequenom scientists sequenced 670 nucleotide bases in and around those four exons. One exon in particular provided graphic evidence of the MassArray's ability to differentiate heterozygous from homozygous alleles (gene variants) in a single high-resolution sequencing reaction. In the mass spectrographic data image, a singleton peak denoted a homozygote (identical inheritance from both parents), while a doublet peak identified a heterozygote (unmatched inherited genes).
Step By Step: How It Works
To acquire this data, the co-authors took DNA from 10 unrelated individuals, in which they detected three zygotic genotypes, and spotted the mutation of a G nucleotide to a C. "We could diagnose the allelic zygosity of those gene fragments from the peaks," O'Donnell pointed out, "What they show is the molecular weight of the diagnostic product. If it's a homozygous allele, you only have one peak. In the heterozygous case, you end up with two different diagnostic products.
"Normally," she continued, "on an electrophoretic gel, these two genetic types can't be resolved, because they're the same size. All you're doing on the gel is separating them by size, which isn't very precise. But mass spectrometry separates them by molecular weight, which is totally accurate, so you can completely identify them as two different species."
Pinning down the individual alleles is work still in progress. "We've actually done some quantitative tests," O'Donnell said, "and we can call the tissue sample as being, say, a certain percent mutated, just based on relative peak height. But we haven't explored that allelic analysis fully yet."
Fine-tuning detection of cancer-causing gene mutations for diagnosis is only the beginning of Sequenom's DNA MassArray story, albeit the most immediately potential application.
The company's president and CEO, Hubert Köster stated, "As our automated, chip-based system becomes available this year, MALDI-TOF mass spectrometry could become more widely used than any other method in drug development research." Köster is senior author of the paper in Nature Biotechnology.
The technology involves three basic steps — DNA preparation, sequencing and analysis.
"First biopsy your tumor," O'Donnell recounted. "Extract the DNA, put it in the well of a microtiter plate, perform amplification by PCR, hybridize the sequencing primer, and carry out the Sanger sequencing right there in the plate.
"What you've generated in the well," she continued, "is your diagnostic product, of which a small sample is put in the chip — by means of that pinhead — for analysis in the mass spectrometer. The chip — 'SpectroChip' in Sequenom's proprietary parlance — has its own wells impressed into its silicon surface.
Microchips Climax Process
"Those wells," O'Donnell observed, "are usually 800 microns in diameter, so you analyze that small spot in the mass spectrometer. You move the chip so each different well is exposed to the laser serially. So say you have 100 spots; it looks at the first one, the DNA gets ionized, then separated by its mass-to-charge ratio. Once that's done — in less than a second — you move to the next spot."
Having spent a lot of years debugging and patenting its DNA MassArray technology, Sequenom is now planning its commercialization strategy.
As a first foray into the marketplace, O'Donnell said, "what we're looking to do is license the technology to collaborators. We're developing an automated process line in-house now, so once you've extracted your DNAs from the patient sample, basically you will put that sample on one end of a modular process line and let it run. What you end up with is your result, which says whether or not it was mutated — or whatever the case might be that you're looking for. No technician interface at all."
She went on: "Actually, our first product will not necessarily be selling the process line, because we're collaborating with people on that. We will actually be selling the support for it, which is to say, microtiter plates filled with the specific reagents, and the SpectroChips. Customers will not provide their own mass spectrometry instruments."
Rather, the company signed an agreement early last year with Bruker-Franzen Analytik GmbH, of Bremen, Germany, to develop an instrument dedicated to analyzing Sequenom's SpectroChip functional microarrays in a high-speed format. (See BioWorld Today, Jan. 14, 1998, p. 1)
"So you'll be able to load up, say, a dozen, SpectroChips in the source at once," O'Donnell concluded, "and analyze thousands of samples per day without even having any interaction with the automated system." (See BioWorld Today, Feb. 18, 1998, p. 1.
Biochemist Bruce Eaton, vice president of chemistry at NeXstar Pharmaceuticals Inc., of Boulder, Colo., is familiar with Sequenom's DNA MassArray program.
"It will be better than gel electrophoresis," he told BioWorld Today, "because of the fact that you can do a larger array more easily. Their figures for mass-to-charge are really quite wonderful.
"I think that when the arrays get large enough," he added, "and as genomic sequencing offers more and more targets, this will be useful in a human diagnostic setting for human pattern recognition, and then correlation to the human disease state. Clearly, there has to be more work done to get it there.
"But these are just technical hurdles," Eaton concluded. "I'm quite confidant they're going to get there." *