“We believe that this could be a solution for smaller, more flexible projects.”
Asper Licenses New SNP-Genotyping Method from Estonian Biocentre
TARTU, Estonia — Asper Biotech, an Estonian biotechnology company, recently obtained a license to commercialize a new method for SNP-genotyping called arrayed primer extension-2 from the Estonian Biocentre.
Asper now intends to begin offering the so-called APEX-2 method as part of its genotyping services, according to a company official.
CEO Alo Merilo told BioArray News this week that the company, based here, attained a non-exclusive license in February to commercialize the technology, and optimized the technology to work on its existing lab equipment last month.
The method is predicated on an early method for lower-multiplex SNP-genotyping called APEX-1 that was also developed at the Estonian Biocentre and licensed to Asper. However, whereas APEX-1 allowed users to develop their own genotyping assays in-house and survey up to 10 SNPs on an array, APEX-2 enables users to do flexible, custom genotyping of up to 1,000 SNPs.
According to Merilo, the flexibility of the method, its relatively low cost, and its increased throughput could benefit users involved in low- to mid-volume genotyping projects while supporting Asper’s ambitions to move into diagnostics.
Asper is active in several areas of genotyping. It sells a series of disease-specific genotyping chips, with an emphasis on eye-related diseases. The company also sells an internally developed array platform called Genorama, activated slides, and offers custom genotyping services.
The company also has a nascent diagnostics business serving laboratories in northern Europe, and is considering branching out into the consumer genetics market. The R&D side of the diagnostics business is currently weighted towards ophthalmic research programs, but Asper also has interests in the oncology, cardiology, and neurology areas.
In terms of consumer genetics, Merilo said Asper is more interested in focusing on ancestry or nutrigenomics, rather than looking to compete with companies like 23andMe or Navigenics that offer more health-focused services. He said the company is also looking to forge strategic partnerships related to Genorama as it focuses more on genotyping and diagnostics.
In both areas, APEX-2 is another tool Asper can use in its service when deciding on the right approach to customers’ projects. “APEX-2 is better than APEX-1 in that, for certain marker considerations, it is a more cost-effective way of doing custom genotyping, hence we were interested in gaining access to that technology,” said Merilo. “It’s ideal if one has a need for a custom-genotyping solution [ranging] from 50 to 1,000 SNPs,” he said.
To be sure, researchers can pursue a number of avenues for lower-multiplex genotyping projects. Illumina, for instance, can move users of its high-density whole-genome genotyping arrays down to its more flexible GoldenGate assay, where they can customize arrays with up to 1,534 SNPs. The company also offers its digital microbead-based BeadXpress Reader for lower-multiplex projects.
Affymetrix, meanwhile, recently acquired the startup True Materials for $25 million. According to Affy, True Materials can survey from several to 10,000 markers in an assay (see BAN 7/29/2008). And there are also custom genotyping options available from companies like Sequenom or Luminex.
According to Merilo, one advantage of APEX-2 over rival technologies is that the company can reach users who might be turned off by the option of investing in a new system just to run custom genotyping assays on an evolving panel of markers. He did not provide pricing for the service.
“We do most of our work in-house and if someone, whether it’s for research or clinical diagnosis, has enough time to collect 200 or 500 samples, that clinical researcher or practicing physician is better off sending those samples to a larger genotyping company who can run them all through in one go and it will be much cheaper,” Merilo said.
“But when the genetic condition is rare enough or conditions require markers to be changed quite quickly, then Asper is the one to call,” he said. “APEX-1 and APEX-2 allow you to do so at a much lesser cost than doing it [using] Illumina or Sequenom or Affymetrix.”
“If you want to do 1,500 SNPs, some companies ask about $80,000 just to develop the assay,” said Andres Metspalu, head of the gene technology group at the Estonian Biocentre and Asper's chief scientific advisor. “In our case, we can do 1,500 without paying $80,000,” he told BioArray News last week.
“If you have thousands of samples to run, then working with some of the larger players makes sense,” Metspalu said. “But small labs don’t have thousands of samples; they just want to play around and do this and that, and I think APEX is filling this niche.”
Metspalu said that alternative methods provided by Illumina or Sequenom were all viable, but that he believes certain researchers need a more flexible platform for their studies. “I recently visited a lab in Canada [where] they have all these machines, all of these assays, but no one is using them,” he said. “Some are too expensive, others are not reliable. We believe that this could be a solution for smaller, more flexible projects.”
Altogether, Metspalu predicts that APEX-2 would allow Asper to expand its clientele in the genotyping market. “If people would like to test for a new disease, they can approach Asper, which will develop the assay, with customers paying for most of the development,” said Metspalu. “APEX-2 is good for follow-up studies to genome-wide association studies, in particular.”
Nucleic Acids Research
The IP for APEX-2 is owned by the Estonian Biocentre and is currently under review in the US, Metspalu said. Metspalu and other scientists at the institution published a paper in Nucleic Acids Research last month demonstrating proof of principle for APEX-2 [Krjutškov K, et al. Development of a single tube 640-plex genotyping method for detection of nucleic acid variations on microarrays. 2008 Jul;36(12):e75.].
The paper demonstrates 640-plex DNA amplification and detection of SNPs and mutations on arrays via four-color, single-base primer extension. The founding principle of APEX-2 multiplex PCR, according to the paper, requires two oligonucleotides per SNP or mutation to generate amplicons containing the position of interest.
The same oligonucleotides are subsequently used as immobilized single-base extension primers on a microarray. The method requires two primers, two spots on the microarray, and a low-cost, four-color detection system for the targeted site, the paper states.
The authors validated the method by comparing call rates for 173 SNPs from 19 samples generated by APEX-2 with Illumina’s HumanHap 370-Due chip. The samples were provided by the Estonian Genome Project, now an institute at the University of Tartu with a biobank of 22,000 samples.
According to the paper, the older APEX-1 method comprises four steps: a) targeted DNA amplification; b) fragmentation and array-based hybridization; c) enzymatic single-base extension on the array; and d) signal detection.
Lead author Kaarel Krjutškov told BioArray News this week that the biggest difference between the two methods is that the APEX-2 uses universal primer tags and two separate PCR phases. The first phase PCR is used for primer specific binding on genome and the amplification of the target region.
“This is the only PCR used in APEX, and because of the long amplicons generated, the PCR product also needs fragmentation,” he said. In APEX-2, the first phase is followed by a second PCR phase with universal primers, and “owing to this stage, one can avoid fragmentation of the products,” he said.
Krjutškov also said that APEX-2 can be used with different detection systems. “We have used single-base extension on [aminosilane with linker]-activated microarrays and a 4-channel Genorama detector like in the APEX method,” he said.
“But if amplicons are generated using the APEX-2 principle, you can make the final detection whichever way you prefer, [whether by] single-base extension in liquid phase, on different solid-phase microarrays, using variants of terminator nucleotides, dyes, and so on.”