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IDT Presents Data on RNase H2-Dependent PCR Genotyping Products at AACR

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NEW YORK (GenomeWeb) – To address the broader needs of genomics researchers, Integrated DNA Technologies has begun offering one of its technologies as a standalone product. The firm presented performance data for its RNase H2-based multiplex qPCR technology this week at the American Association of Cancer Research meeting in Washington, DC.

IDT has been an established player in the custom oligo market for many years. Founded in 1987 by Joseph Walder in Coralville, Iowa, the firm has also had as a component of its technology portfolio an RNase H2-dependent PCR technology developed by Walder.

Criss Walworth, the firm's vice president of genomics solutions, noted that, "The RNase H2 enzyme has some interesting properties that were known, characterized, and patented by our founder many years ago."

For example, because it was originally discovered in Pyrococcus abyssi — a hyperthermophilic archea from hydrothermal vents on the sea floor near Fiji— it has thermostable properties that enable its use in PCR.

Now, making this chemistry available is also "a real example of the evolution going on within IDT," Walworth said. The company is increasingly looking to offer more value-added products in the genomics space, expanding well beyond the impression that it is simply a custom oligo supplier. "I think the surprise to those who are less familiar with us is that we're actually much more than that," Walworth said.

The new RNase H2-based multiplex qPCR technology is called rhAmp SNP Genotyping. It harnesses the RNase H2 enzyme, as well as a novel, proprietary Taq polymerase that has high allelic discrimination powers, and a universal reporter system to deliver higher signal-to-noise ratio, said Aurita Menezes, a qPCR product manager at IDT.

At AACR this week, the firm presented two posters on the method. One describes use of rhPCR assays in sensitive and accurate detection of germline SNPs and somatic mutations, noting that the high specificity and sensitivity of multiplex rhPCR assays makes the technology suitable for detection of somatic mutations in cancer.

Specifically, according to the poster, the assays contain a locus-specific reverse primer and allele-specific primers, and each primer contains an RNA base and a short, removable, 3′ terminal blocking group. The RNaseH2 enzyme recognizes and binds the duplex region containing the RNA base and selectively cleaves the duplex, which activates the primer.

The rhPCR method can be run in singleplex or multiplex mode using the universal reporter system — consisting of a single, universal forward primer and different probes containing fluorophores — and the proprietary mutant TaqDNA polymerase with high mismatch discrimination allows for selective extension of new DNA strands from the newly unblocked primers.

"Using a single-tube reaction, this assay system can effectively discriminate between closely related sequences that differ by only a single nucleotide," the authors noted.

A second poster demonstrates the rapid detection of mutations in isocitrate dehydrogenase 1 and 2 genes using an RNaseH2-dependent, multiplex quantitative PCR assay. IDH1/2 mutations have been identified in a subset of cancers, and their detection can have diagnostic, prognostic, and therapeutic implications.

In addition to these posters, the firm is also hosting a seminar this week at qPCR dPCR & NGS 2017 in Germany this week, where it will discuss multiplex rhPCR and its applications in amplicon sequencing, and rhAmp SNP genotyping. 

Although there is plenty of competition in this general market, Menezes pointed out that the firm has some distinct competitive advantages.

For example, compared to Thermo Fischer Scientific TaqMan-based assays, IDT anticipates having a lower cost. "For the Thermo-based assays, you have to design two new probes for every new SNP that you want to investigate," Menezes said, and there is an associated cost to that service. Furthermore, the universal reporter system allows IDT to "provide performance at a very affordable price," she said.

IDT also sees KASP assays from LGC as a competing product, but Menezes noted that IDT's long experience with its technology may provide some benefits. "We are harnessing our core capabilities in oligo manufacturing [and] turnaround time is determined by making three allele-specific primers that include an RNA base," she said. "That is our core strength, so we are able to do it quickly, while it is something that others struggle with." She estimated the product will have a turnaround time of a few days.

The firm sees customers for this genotyping product in academia as well as the pharmacogenomics space, testing labs, and core genotyping facilities.

Overall, IDT's business might be broken down into a few main segments, Walworth said. The custom business includes the core custom oligo service, as well as the firm's synthetic biology business, and GMP and third-party manufacturing.

IDT also has a functional biology group which covers its CRISPR products, including the Alt-R System genome editing product. These "have been generating a lot of buzz and have been very well received by the market as well," Walworth said.  

Another business domain is the genetic analysis group, which includes next-generation sequencing products, such as target enrichment products, xGen Lockdown probes and panels, and the firm's exome panel. Walworth noted that the xGen Exome Research Panel was recently the subject of a case study in which the firm contracted an independent third party to compare products from commercial exome suppliers, including IDT. The study showed the IDT exome panel had significant performance improvements over other products, she said, and the firm is seeing this supported by trends in market adoption.

IDT's qPCR portfolio includes its PrimeTime assays, "which are traditional 5' nuclease assays for gene expression and other types of real-time analysis," Walworth said, and the new rhAMP SNP product, as well as "a host of other predesigned assays for intercalating dyes like SYBR and a few other probe design options for qPCR."

The rhAMP product will formally launch later in the spring, and Walworth said the firm is envisioning it as the beginning of a number of additional product extensions.

Because of the characteristics of rhAMP, including "the ease of use and the performance benefits of the combination of RNase H2 and mutant polymerase," the firm sees "the potential impact of this core technology as much broader than just qPCR solutions," Walworth said. Future applications might include sequencing-related products, a subject of previous posters the firm presented at Advances in Genome Biology and Technology meetings, Walworth said.

"The first implementation is genotyping chemistry, but we see this as a technology that has legs, and that can be applied into a number of other areas," she added. "Essentially, at its most basic level, you could think of it as the next evolution of PCR that addresses some of the issues that have plagued PCR for quite a while, such as primer dimers and lack of specificity."

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