German company Analytik Jena has launched a kit for enrichment and extraction of circulating cell-free DNA, which it believes is quicker than currently commercially available products, requiring fewer steps as well as lower starting volumes.
Analytik Jena has long been known as a manufacturer of biotechnology and life sciences instruments, as well as high-end optical consumer products. The company was established 30 years ago, and started its life sciences division in 2006. It is now growing the life sciences product line, and, at the same time, hoping to capture a larger share of the US market.
Ingo Jenneckens, export manager for AJ, told PCR Insider that the company's strategic acquisition last April of UVP, a California-based company that provides digital imaging systems for applications in proteomics, genomics, and plant and animal sciences, should help that cause. "Unfortunately our brand, AJ, is not very well known in the United States so far … for this reason, last year we did a strategic acquisition [of] UVP, so this is a daughter company of Analytik Jena now, and they take care of our products in the United States and Canada. So, I think AJ will also become a brand name in North America very soon," Jenneckens said.
"AJ life science is supplying what we call 'workflow solutions,' so we provide different instruments, starting from homogenizers, automated nucleic acid extraction, liquid handling, PCR, qPCR, gel imaging," he said.
Adding to those workflow solutions, AJ's new cfDNA kit is the first in a "biomolecule enrichment" line to be offered by the firm using a patent-pending polymer-mediated technique.
The typically low abundance of cfDNA makes enrichment important, but also puts a premium on getting the highest yield possible with the least amount of loss. Jenneckens claimed that the cfDNA kit has been tested against "the market leader," and a brochure for the kit compared extractions of free-circulating DNA using AJ’s PME technology versus "a commercially standard extraction kit for free-circulating [nucleic] acids (supplier Q)," showing equivalent results.
While AJ's kit it is too new to have been cited in any publications yet, Jenneckens said the company has gotten good feedback so far from customers, adding, "Customers are highly welcome to challenge our kit [against competitors]."
The cfDNA kit can be used on plasma, serum, urine, other bodily fluids, or cell culture supernatant. In a manual for the kit, the company also compared amplification levels from serum initially collected into nine different types of blood collection systems, and suggested an optimum collection method.
The kit is available in two sizes to perform either 10 or 50 reactions. Jenneckens emphasized that it is supplied with enough reagents to allow each reaction to be run on the maximum volumes. "We really provide the full amount of chemistry so that you can prepare the maximum amount, and this makes the kit also very effective pricewise," he said. The list price in Germany for the 50-reaction kit is €650 ($880), said Jenneckens, making it competitive with the 50-reaction QiaAMP cell-free DNA kit, which is listed at $1,031 on the Qiagen website.
The cfDNA enrichment kit uses a two-step process. Up to 5 ml of plasma or serum, or up to 10 ml of urine, are incubated in the polymer-containing solution and centrifuged. Then, pelleted cfDNA is bound to a spin filter and eluted. The company estimates extraction times to be 30 minutes for a 1-ml starting sample, and one hour for up to 10 ml.
Jenneckens said AJ's faster and simpler protocol is made possible because of the company's patented low-salt buffers, which eliminate a salt-removal step. He claimed these buffers go against the dogma of nucleic acid extraction, which "always has considered that DNA binds to silica at high-salt concentrations, especially chaotropic salts [with] high molarities." He suggested AJ's lower-salt buffers could make enzymatic reactions, such as the proteinase K step, more efficient. "I compare it [to the enzymatic] activity in a fresh water lake versus the Dead Sea. Our chemistry has a low salt concentration, like fresh water, so the enzyme is really highly active, and also I would expect that other enzymes that do not work in other systems might work in our buffer system," he said.
The cfDNA kit has applications in "tumor and prenatal diagnosis, as well as pathological states, including trauma, sepsis, myocardial infarction, stroke, transplantation, diabetes mellitus, and hematologic disorders," according to the company's website.
Though AJ's cfDNA kit is a research-use-only product, evidence continues to accumulate for the utility of cfDNA in cancer diagnosis and treatment response. A recent Clinical Cancer Research study, for example, used Qiagen's cfDNA kit, QiaAmp, to enrich cfDNA from plasma, and ddPCR to measure the resistance mutations EGFR L858R and exon 19 deletion in KRAS-mutant lung cancer. Researchers analyzed whether cfDNA could be a proxy for erlotinib treatment response. The authors concluded, "Serial quantification of plasma genotype allows noninvasive assessment of response and resistance, including detection of resistance mutations up to 16 weeks prior to radiographic progression."
The kit is the first in AJ's polymer-mediated enrichment line, but adds to an already vast catalog of nucleic acid sample preparation kits. The life sciences division manufactures, for example, 12 different kits to isolate genomic DNA depending on sample sources and size of reaction. There are also kits for plasmid DNA, microbial DNA and RNA, total RNA, as well as kits for different PCR clean-up needs.
Jenneckens anticipates the next to launch in the PME line will be a kit developed for epigenetic analysis. It will include a bisulfate conversion reagent, "which has been shown to have very low impact on the DNA integrity," Jenneckens said, adding that this reagent showed high yields as well as "very low false positive and false negative rates, so really highly efficient conversion rates."
The firm also plans to market another cfDNA extraction kit to run on their automated system called InnuPure, in which the pellet obtained after enrichment is loaded as the sample and extraction is performed automatically. In addition, AJ recently debuted a microbiome extraction kit called Looxster, Jenneckens noted, to selectively enrich fungal and bacterial DNA from preparations with high fractions of eukaryotic background DNA.