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In Quest for Universal Microarray, EraGen Tags McGill Univ. as a Partner


EraGen Bioscience and McGill University have jointly embarked on a quest for the perfect universal microarray, joining a select group of scientists who hope to eliminate the need for sequence-specific probe sets.

EraGen, a Madison, Wis., bioinformatics firm, is seeking through the collaboration to combine its expertise in designing oligonucleotides with that of McGill researcher Michael Hallet, a professor at the university’s School of Computer Science. The group will develop algorithms for arrays not specific to a single organism or set of genes, which will allow for simultaneous monitoring of multiple hybridizations, according to EraGen.

“Last year we created our first version of the algorithm, put it through its paces, and came up with a set of tags that worked very well,” said Chris Scherrer, head of probe design for EraGen. “What Dr. Hallet gives us is vast experience with combinatorial methods for developing non-redundant strings of DNA that we can use to make a new tag set.”

The tags form the foundation of the technology that enable microarrays to be “universal”. Instead of gene-specific oligonucleotides that are 25 or more bases long, the oligonucleotide probes affixed to this “universal array” are nine or ten bases long. These probes comprise tags. These tags are designed along with complementary short strands called anti-tags.” The anti-tags are added to the target analyte mixture during the amplification process. Then these analytes, which are also tagged with fluorescent biomarkers, will hybridize to the tags at their anti-tag end, producing a fluorescent signal.

“A universal chip allows you to put a small piece of DNA on the chip, then pull out a mixture of amplified or expressed DNA that has the anti-tag to what’s on your chip,” said Scherrer.

Ordinarily, such short tags would produce a major problem with random hybridization. But EraGen has engineered these oligonucleotides to hybridize only to the complementary tag or anti-tag by adding two synthetic bases to the four nucleotide bases used in “natural” DNA. The two extra bases, isoC and isoG, are utilized by common DNA-modifying enzymes, so they can work within a standard hybridization protocol. The oligos are manufactured using the company’s proprietary automated gene synthesis process, entitled AEGIS “An Expanded Genetic Information System.” The company uses bioinformatics to select the sequences for the short oligonucleotides.

Currently, EraGen uses polystyrene beads as substrates for its tags, but said a glass slide could be used as well.

In the McGill collaboration, EraGen plans to extend the existing algorithms that utilize the AEGIS bases to create these tags. While the current algorithm enables users to probe for 100 different analytes simultaneously, the next generation that Hallet is helping to design aims to provide thousands of different probes per array system. The partners are also developing software that uses a thermodynamic model to detect DNA secondary structure.


“Universal” Competition

Several other efforts have been mounted to develop universal array systems.

Tm Biosciences, a Toronto-based startup, just began marketing the Tm-1000 Universal Sequence Set at the end of November. Like the EraGen system, this set consists of complementary tag/anti-tag oligonucleotide pairs, one of which attaches to the target analyte and the other of which is secured to the array surface. The company also uses a bead platform, the Luminex LabMap technology, as a substrate.

Unlike EraGen, however, Tm Biosciences uses 24-mer oligonucleotides made of the four natural bases in DNA. Instead of linear moieties, the company has experimented with designing its probes as hairpin structures in order to minimize cross-hybridization. Tm also employs its own algorithm, the Sequence Design Engine, which uses pattern matching and similarity thresholds, along with DNA thermodynamics, to select the oligo sequences.

In company literature, Tm scientists have reported that their system has a very low level of non-specific hybridization, with only 6 out of 9,900 possible non-specific hybridizations occurring in an experiment where 100 tag/anti-tag pairs were hybridized. These non-specific hybridizations generated signals that were no more than 10.2 percent as strong as the specific hybridizations, allowing them to be easily isolated as artifacts.

“The sequence set combines high specificity with utmost flexibility,” the company literature says.

Another startup, New Haven, Conn.-based Agilix, is developing universal microarrays based on the patent inventions of Yale professor of pathology Paul Lizardi. Lizardi, who invented rolling circle amplification (RCA) in 1995, has patented a system that uses RCA in an effort to detect gene expression patterns in all genes from any organism. This system, the “fixed address analysis of sequence tags” (FAAST) transcription analysis system, uses the same set of universal probes on each array.

In the system, the tag-like molecules, which are either partially or totally composed of nucleic acids, are associated with the target molecules. Next, these combination molecules are amplified using rolling circle amplification. Finally, the detection stage involves multicolored coding probes that allow simultaneous detection of different target circles.

Agilix has licensed the patent to the FAAST technology, US Patent Number 6,261,782, which was granted to Lizardi in July. This past week, Lizardi received number 6,329,150 “Unimolecular segment amplification and sequencing.” Agilix is likely to license this technology as well.

The Agilix platform currently is the only one that includes rolling circle amplification, a method that many believe involves less amplification bias than other methods such as PCR. The software algorithm used to design the probes in Lizardi’s operation is also more high-throughput than others, enabling the user to program up to one million addresses per experiment.

Agilix is now offering the system under special early-access agreements.

But while Agilix may currently offer the advantage of throughput, and Tm actually has a product on the commercial market, EraGen claims that it has the ultimate edge in offering protocols that are more convenient than those of its competitors. Its tag/anti-tag experiments can be done at room temperature with minimal washing steps, while others require temperatures of 45 to 50 degrees Celsius, said Scherrer.

EraGen is planning to sell its universal array system either on its own or in partnership with another collaborator. “We are looking for any sort of partnership that would help us to exploit the advantages of our system in the scientific community,” said Scherrer.


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