NEW YORK (GenomeWeb) – Researchers from Taiwan have taken an important step toward potential clinical use of their novel PCR enhancement method that involves using a peptide nucleic acid clamp (PNA) molecule not only to inhibit amplification of wild-type DNA, but also as the sensor probe for a particular assay.
In a new study in Cancer Genomics and Proteomics, they used their PNA probe assay to analyze 49 melanoma samples and correctly detect three types of mutations in 17 samples, with better sensitivity than conventional PCR plus Sanger sequencing.
PNA molecules can be designed to clamp onto wild-type alleles, preventing PCR synthesis of those molecules and promoting highly sensitive PCR amplification or other detection of a target mutation even amidst a large background of wild-type DNA.
The authors of the new study — led by Chiuan-Chian Chiou, a researcher at Taiwan's Chang Gung University — are not alone in their desire to advance better molecular detection methods based on PNA clamps.
For example, researchers from Italy have reported on their use of PNA clamping in PCR assays for genetic mutations in the hematology and oncology fields.
An international team also published a study in 2014 showing that PNA clamping PCR was significantly more sensitive than Sanger sequencing in detecting KRAS mutations in both tumor tissue and plasma samples.Â
Still other researchers have explored coupling PNA clamps with electrochemical detection chips to avoid PCR's enzymatic amplification process.
Diagnostics start-up DiaCarta has engaged another clamping technique it calls QClamp — which uses xenonucleic acid, or XNA — to develop a set of commercial real-time PCR assays. The company received CE marking in 2014 for several kits that use the method to detect various cancer-related mutations, including KRAS, in whole blood samples.
More recently, it expanded access to these tests to the US after its California-based lab became CLIA certified to provide clinical testing services using QClamp.
The Chang Gung University team's marriage of clamping and target detection using only PNA is unique though, Chiou told GenomeWeb in an email. The team believes that this ability to design assays with a single primer and probe set can reduce cost and simplify operation compared to other methods.
"To our knowledge, we are the only group that uses this design," he wrote.
In Chiou and his colleagues' method, which they first published in 2006, a fluorophore-tagged PNA molecule serves as both the PCR clamp and sensor probe, which inhibits the amplification of wild-type templates during PCR and also marks multiple types of mutant signals during melting curve analysis.
"PNA can bind to both the wild-type and the mutant templates but generate different melting peaks," Chiou explained in his email. "Once the PCR condition has been optimized to inhibit wild-type amplification completely, only [the] mutant peak appears [and] through this featured melting peak, our method can eliminate false signals from non-specific PCR products and has better specificity," he said.
In contrast, some PNA or XNA probe-based methods, like DiaCarta's, use SYBR green I to report mutant PCR products. According to Chiou, because SYBR green I lacks selectivity between real mutants and nonspecific PCR products of primer dimers, it can pose assay specificity problems.
Meanwhile, some other methods have been developed that combine both PNA and locked nucleic acid (LNA) probes. But, because PNA and LNA have the same binding site, these probes compete with each other, necessitating design of specific LNA reporters that are complementary to a particular stretch of mutant DNA.
The downside of this is that if more than one type of mutation occurs in the target region, several different LNA probes have to be synthesized and then tested for their efficiency and compatibility when combined in the same reaction, Chiou and his colleagues have argued.
In their study in Cancer Genomics and Proteomics, Chiou and his team initially demonstrated the sensitivity of their single-probe PNA method by using mixed template samples consisting of different mutant DNA ratios in a wild-type background.
"Under optimal PCR conditions, the wild-type DNA amplification was completely inhibited, and only the reactions containing detectable mutant templates generated a melting peak," the authors wrote. Moreover, they could detect BRAF V600E mutations at levels as low as 0.05 percent with marked mutant melting peaks. "In contrast, the reaction containing only the wild-type template generated no peaks," they said.
Then, the researchers applied their assay to clinical melanoma specimens — 49 formalin-fixed paraffin-embedded samples — comparing the results to traditional PCR plus Sanger sequencing.
The conventional method detected mutations in 11 samples, including 10 V600E mutations and one V600K mutation. In contrast, the PNA probe assay detected mutations in 17 samples, including all those detected by the conventional method plus an additional six mutations.
To verify the results of the PNA probe assay, the team also sent PCR products from the assay for Sanger sequencing, which clearly revealed the accurate mutation signals.
According to Chiou and his colleagues, their PNA probe assay can be completed in a single tube on a standard real-time PCR instrument.
Although different mutations have different melting profiles, as evidenced by the team's BRAF mutation study, the resolution of these curves is "generally not good enough to distinguish each specific mutation type," the authors wrote.
"Fortunately, the PNA probe assay only enriches the mutant templates but does not change them," they explained, which means mutant PCR products can be further sequenced to determine the different mutations types present.
Chiou said in his email that the group has now developed assays for EGFR and PIK3CA mutations in addition to BRAF, and is hoping to make the method commercially and clinically available. The team is looking into technology transfer options, and also would welcome marketing partners, he said.
Although the team's recent study was on FFPE tissue specimens, it could also be useful for liquid biopsy applications, the group concluded.
"In body fluids, disease-derived nucleic acids [are] rare compared to normal DNA. The presented PNA probe assay has good sensitivity and may serve as a powerful tool in the application of liquid biopsies," the authors wrote.