Enzo Biochem will later this year debut a next-generation version of its branched DNA signal amplification technology that it hopes to make available to microarray and fluorescence in situ hybridization users.
Elazar Rabbani, founder and CEO of the New York-based life sciences firm, told BioArray News last week that the firm has overcome limitations in its existing bDNA amplification technology that should make it more attractive, particularly for molecular diagnostics use.
"Branched DNA is [an] extremely powerful signal amplification" approach, said Rabbani. "Its limitation is that bDNA has to be delivered through nucleic acid." To date, bDNAs have been designed to connect to a specific target, according to Rabbani. As arrays can contain millions of targets, it has not been feasible to couple them with a bDNA-based signal amplification approach.
"You cannot apply bDNA directly on an array because of the … corresponding connections," said Rabbani. "That is why nobody has applied bDNA in that format. Each target requires a specific connection, and that is very expensive."
According to Rabbani, Enzo has overcome this limitation through "complex chemistry," providing a "universal approach" to bDNA-based signal amplification that does not rely on a nucleic acid delivery system. He declined to further describe the complex chemistry behind Enzo's approach.
While Enzo first plans to make the next-gen platform available for FISH, it "absolutely could become available in an array format," said Rabbani. "That was one of our objectives in developing this," he said. "We already do produce reagents for array assays that rely on streptavidin conjugates, so we will just be changing the streptavidin to bDNA," he said.
As for when it could become available, Rabbani said that Enzo hopes to "push the project forward this year," but cautioned that "production and validation take time."
The new technology should be able to work as well for DNA as for RNA samples, which could benefit researchers who use arrays for gene expression profiling. "There are challenges in RNA evaluation," said Rabbani. "When you have small samples the challenge is how much to amplify the RNA. Our dream is to be able to assess profile expression better by reducing ambiguity from amplification."
The advantage of bDNA, Rabbani claimed, is that it amplifies the signal, as opposed to the target. "Everyone wants quantitative analysis of RNA, but [target] amplification interferes with the quality of that number," he said. "The more sensitive [signal] detection you have, the better off you are."
FISH Meets bDNA
While bDNA-based signal amplification kits for arrays are on the horizon for Enzo, the firm has more concrete plans for applying its bDNA platform for FISH, replacing the "very large" fluorescently labeled DNA probes that are currently used to generate signals for chromosomal analysis, Rabbani said.
However, these large probes contain primarily non-unique sequences that will "cross-hybridize indiscriminately over the entire chromosomal range." In order to prevent this non-specific signal generation, the labeled probe additionally contains a large amount of unlabeled repetitive DNA in order to "mask" hybridization of the labeled probe to the non-unique sequences, he said.
One way to avoid this addition is to isolate probes that lack the non-unique repetitive sequences. However, probes consisting of only the unique sequences are very small in size, said Rabbani. Since the unique sequences occur as small islands embedded in a sea of repetitive DNA, the similarly small probes generate specific signals that are difficult to detect by standard methods.
According to Rabbani, Enzo has developed bDNA fluorescent probes that can detect small chromosomal targets in a cell.
"As a model for this proprietary system, we have tested biotin-labeled HPV16 probes in a cell line that contains a single integrated copy of an 8-kilobase viral genome with consistent and reproducible detection," he said. "Any target of this size in a chromosome should be readily and specifically detectable in a cell."
For FISH, Enzo's bDNA platform allows the use of smaller, less expensive probes; eliminates the need for adding repetitive suppressor DNA; and enables the detection of small-scale rearrangements and deletions, Rabbani said.
He added that the technology functions as a "universal reagent that can detect any biotinylated probe, including antibodies as well as DNA," Rabbani said. Enzo's bDNA technology can also be used to detect the integration of viruses into chromosomes, which has been theorized to be a marker for progression to cancer.
Enzo's established bDNA assays already compete against a number of offerings, depending on the application. Affymetrix, for instance, provides a menu of Panomics bDNA-based products. The Santa Clara, Calif.-based firm describes its bDNA assay as a "sandwich nucleic acid hybridization method that uses bDNA molecules to amplify signal from captured target RNA."
Jasmine Gruia-Gray, Affy's vice president of global marketing, told BioArray News that "Panomics products aren't used in molecular diagnostics but we do have products in molecular pathology, [and] translational and cancer research.
"Due to [US Food and Drug Administration] regulations, we’re not able to make any diagnostics claims," she said. Gruia-Gray declined to comment on Enzo's offering.
Another competitor is Siemens, whose automated Versant 440 Molecular System relies on bDNA chemistry to perform HIV and hepatitis viral load monitoring. Other rivals include Chiron and Bayer Diagnostics, which offer similar bDNA-based tests for viral testing.
Enzo's competitors in the area of bDNA array labeling will be any firm that provides array labeling kits, such as Kreatech Diagnostics, Life Technologies, and Qiagen. Of these, Amsterdam-based Kreatech will likely be one the most direct competitors. The company sells array labeling kits and FISH probes based on its universal linkage system technology.
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