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DNA Clutch Probes Make Electrochemical Clamp Assay Applicable to Circulating DNA


NEW YORK (GenomeWeb) – Expanding upon its earlier development of an electrochemical chip method to detect circulating nucleic acids, a team from the university of Toronto has taken an important new step that allows its approach to sensitively detect not only RNA molecules but also target DNA sequences directly from samples without enzymatic amplification.

The new advancement, which the team published earlier this month in the Journal of the American Chemical Society, was enabled by a technology called DNA clutch probes.

Using these probes, lead investigator Shana Kelley and her coauthors were able to detect cancer-associated DNA mutations in serum samples from patients with lung cancer and melanoma using their previously developed electrochemical chip platform.

The group debuted that technology, which relies on clamp molecules to isolate and bind to wild-type or non-target mutant DNA,  in a 2015 study in Nature Chemistry. In that work, the researchers showed that they could detect target sequences directly from patient samples without the need for enzymatic amplification, and with the same sensitivity as a comparable PCR method.

Electrochemical sensors for nucleic acids offer advantages over PCR because of their potential to be much more rapid, user friendly, and cost effective. Meanwhile, direct detection from clinical samples like serum means an assay can avoid the loss of sensitivity or biasing of results by sample-prep methods needed to purify or clean up samples for enzymatic detection.

However, while Kelley and her colleagues were able to demonstrate that their clamp molecule-based electrochemical chip could detect target circulating nucleic acid sequences with at least the same sensitivity as PCR, they found that the method could only detect circulating RNA, not circulating DNA mutations.

In their new study, the investigators overcame this limitation using DNA clutch probes, which are pairs of single stranded DNA molecules that bind to a particular target in denatured DNA.

Kelley told GenomeWeb in an email that these probes are designed to pull cell-free DNA apart so that the single-stranded form is stabilized.

"The clutch probes are complementary to a sequence that neighbors the one that is the detection target, and are designed to have thermodyanmic properties so that they don’t compete with the binding of the target to our electrochemical sensor," she explained.

The clutch probes prevent DNA strands from reunifying, essentially holding on to the complementary strand and leaving the remaining single stranded DNA able to hybridize with a detection probe. Then the group's previously described clamp molecules hybridize to the single-stranded wild-type DNA in the sample, which means that the only thing left that can hybridize to the electrochemical chip sensors is the single-stranded mutant DNA.

In their study, Kelley and her team validated this approach first using a set of KRAS mutation-positive and normal control samples combined with different components of the assay —  either only clamp molecules or clamps and clutch probes together.

As expected, they only saw electrochemical signal changes for the samples of ctDNA from a mutation-positive patient in the presence of the clutch probes.

When the group further experimented with samples with different concentrations of KRAS mutant DNA in a background of wild-type, they found that the approach was able to detect mutations down to a frequency of 0.01 percent, which matches or exceeds the best LODs achieved by PCR-based detection methods.

Finally, the team applied the approach to a set of serum samples from nine lung cancer patients and nine melanoma patients with known mutation status, comparing the results to clamp-based PCR.

"We were able to show that we could generate the same results analyzing patient cfDNA as PCR," Kelley wrote in her email. In fact, the electrochemical chip actually correctly identified a BRAF-mutant melanoma sample that clamp PCR couldn't determine.

Kelley said that she and her colleagues are now working on analyzing a larger set of patient samples.

Although the team has not yet had made any solid commercial plans around the technology, Kelley said that aside from the cancer mutation applications there is also a possibility that the platform could be useful in the context of diseases caused by DNA viruses.

The team hasn't tested this yet but is planning to, she said.