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UMDNJ Group Develops 'Super' Selective Primer Tech for Low-Level qPCR-Based Mutation Detection


SAN FRANCISCO — Researchers from the University of Medicine and Dentistry of New Jersey and New Jersey Medical School this week unveiled a new qPCR-based mutation-detection chemistry at Cambridge Healthtech Institute's Molecular Medicine Tri-Conference held here.

The technology, called SuperSelective Primers, was developed in the laboratory of Fred Kramer, a researcher at UMDNJ, NJMS, and the Public Health Research Institute Center, which is affiliated with the two medical schools.

The primers work by amplifying mutant sequences that are perfectly complementary while virtually ignoring wild-type sequences that are mismatched by just a single nucleotide, and they can detect and quantify as few as 10 mutant target molecules in the presence of 1,000,000 wild-type molecules.

Kramer presented his lab's technology as part of Tri-Con's quantitative real-time PCR symposium. During his presentation, Kramer noted that a major goal in the molecular diagnostics space is to develop sensitive and specific ways to detect and quantitate extremely rare cancer cells by identifying somatic mutations in clinical samples containing very abundant normal cells.

He cited recently established commercial technologies such as Seegene's dual priming oligonucleotides and Swift Bioscience's MyT primers as examples of highly selective assay chemistries. In general, Kramer noted, technologies such as these make use of very small primers – perhaps six or seven nucleotides long – to increase selectivity.

"The problem with using a little primer is that a sequence seven [nucleotides] long appears in many different places in the human genome," Kramer said. "You can't just use that; it will prime all kinds of products. You need a way to get that primer to just bind to the place in the human genome [with the] gene you want."

DPO primer technology solves this specificity issue by linking the short primer to an anchor sequence. The SuperSelective primers, which Kramer was publicly disclosing for the first time, draw from the same well of inspiration.

Generally speaking, the SuperSelective primers operate by using an "anchor sequence" that provides selectivity and can be "permissive" if necessary; a "foot sequence" that contains an interrogating oligonucleotide, which itself is most often at the 3' penultimate position; and a "bridge sequence" opposite an intervening sequence in the target, which creates a "bubble" in the construct.

"The idea is to add to your primer an additional sequence at the 5' end called an anchor sequence, which is a traditional sort of primer in that it is long enough, and at the annealing temperature … will only bind to the desired gene," Kramer explained. "Then we connect that anchor sequence to the primer sequence by something called a bridge sequence."

This bridge sequence, he noted, is not complementary to a corresponding intervening sequence in the template. The end result is that "basically these primers go to the right gene, but then quite free and separate is this … little primer, which will form a primer with the mutant you want to detect, but because of the mismatch will not form a primer with a wild-type that you don't want to detect," Kramer said.

The initial constructs that Kramer's lab has produced comprise 24 nucleotides in the anchor sequence, a 14-nucleotide long bridge, and a seven-nucleotide long primer, in which the interrogating nucleotide is in the penultimate position next to the 3' end.

"We call this a 24-14-5:1:1 primer – 24 nucleotides in the anchor, 14 in the bridge, five that are perfectly complementary to both mutant and wild type; one that is the interrogating nucleotide that is only complementary to one of them; and a last one which is complementary to both," Kramer said.

Kramer noted that the SuperSelective primers are used in exactly the same way as primers in a normal PCR reaction, with the same concentrations and conditions.

"One of the wonderful things about these primers is … if [the SuperSelective primer] is able to initiate chain elongation, and you get an amplicon, that amplicon is … exponentially amplified in the usual way," he said.

Kramer's lab has tested the primers to detect extremely low-level mutant sequences in clinically relevant genes, such as EGFR and BRAF, and has demonstrated that the synthesis of mismatched wild-type target molecules in the sample is suppressed enough to enable the detection and quantitation of as few as 10 molecules of perfectly complementary mutant target molecules in the presence of 1 million wild-type target molecules – a ratio equivalent to or better than most any commercial assay.

The lab is still trying to determine the exact mechanism of action of their primers, but believes that their selectivity is due to a combination of the "lower abundance of mismatched hybrids," due to their shorter length, as well as "the shorter … mean persistence time of mismatched hybrids, which makes it more difficult for a DNA polymerase molecule to find a hybrid to form a stable complex."

A SuperSelective primer-based reaction can be performed using any existing qPCR instrumentation platform, Kramer said. He also noted that the researchers have successfully used the primers with a SYBR Green probe-based detection method, as well as molecular beacons, a hybridization probe technology that Kramer's lab also developed.

The group has applied for patents on the SuperSelective primer technology but has not yet published on it, Kramer noted. The lab has also applied for a grant to explore multiplexing with the technology and to begin applying it to clinical samples.