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Riken, DNAForm Team Develops 'Eprobes' for Combined Real-Time PCR, Melt Curve Analysis

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A team of scientists from Japan's Riken and DNAForm has developed a highly efficient fluorescent molecular probe for combined real-time PCR monitoring and melting curve analysis, according to research published this week in PLoS One.

Called "Eprobes," the technology has demonstrated enhanced background signal reduction and DNA-binding affinity, as well as low false positive rates, and should enable both quantitative and qualitative analysis of PCR amplicons on standard real-time PCR instruments in a single consecutive procedure, according to the study's authors.

As such, the Eprobes may empower new types of highly sensitive and specific molecular assays for gene expression analysis, determination of genomic copy number variation, and allele-specific discrimination of SNPs or somatic mutations, among other applications, they said.

In addition, DNAForm, a 1998 Riken spinout formed to help commercialize biotechnology developed at the institute, holds a license to the Eprobe technology and is seeking to commercialize both research kits and molecular diagnostic products based on the probes, PCR Insider has learned.

According to the scientists, intercalating dyes like SYBR Green are very sensitive but prone to primer-dimer formation and other PCR artifacts, while hydrolysis probes like TaqMan probes, Molecular Beacons, or HybProbes are desirable for highly specific PCR reactions but can be complicated to design and do not enable combined monitoring of the amplification reaction and melting curve analysis.

The Riken and DNAForm researchers had previously designed and disclosed a technology to address these issues: exciton-controlled hybridization-sensitive oligonucleotides, or ECHO, probes. These probes commonly possess a modified thymine carrying two fluorescent dye moieties, and demonstrate strong background suppression involving just a single modified nucleotide, allowing for easier synthesis and design.

These ECHO probes have been used in multiplexed amplification reactions using processes such as isothermal amplification, or for fluorescence in situ hybridization or RNA detection in living cells.

However, in order to use the ECHOs as hybridization probes in real-time PCR and melting curve analysis, the scientists modified them into Eprobes, which are blocked at the 3' end to prevent primer extension. Thus, the probes do not interfere with amplification reactions, but act as independent hybridization probes in signal detection and amplicon analysis.

In an email to PCR Insider, Kengo Usui, a researcher with Riken's Center for Life Science Technologies Preventive Medicine and Diagnosis Innovation Program, and a co-corresponding author on the paper, noted that while the fluorescent dyes on Eprobes are quenched by themselves in a non-hybridization state, quenching is arrested and the dyes are able to fluoresce only during target-specific hybridization.

"For example, free double-stranded DNA intercalating dyes [such as] SYBR Green … not only intercalate target-specific amplified product but also misamplified product such as primer-dimers in a PCR reaction," Usui said. "Therefore, Eprobes have activity to reduce background signal."

Additionally, in contrast to TaqMan probes, Eprobes should not be digested during PCR, keeping the intact probe available for melting curve analysis after completion of PCR, according to the researchers.

They tested the Eprobes as hybridization probes in real-time PCR and melt-curve assays using as templates exon 21 of the human EGFR gene, and compared them to both SYBR Green- and TaqMan-based assays. All PCR reactions using the Eprobes yielded DNA fragments of the correct 229-bp length, indicating that the 3' end blocking group in the Eprobes indeed abolished primer extension.

Furthermore, the Eprobes enabled real-time PCR monitoring and provided similar Ct values to a TaqMan probe, and higher Ct values than those observed using SYBR Green.

Next, the scientists tested the ability of the Eprobes to distinguish between the wild-type and mutated allele of exon 21 in EGFR by performing asymmetric PCR and subsequent melt curve analysis. The wild-type and mutated allele could be clearly distinguished by their different melting temperature values, with the point mutation reducing the TM value of the wild-type probe within the expected range.

Finally, the researchers confirmed that Eprobes having two different dyes can be used in multiplex PCR in the same way that the scientists demonstrated in isothermal amplification reactions using a previous version of the technology. In this case, they amplified regions of the EGFR and KRAS genes from genomic DNA with specific primer sets and Eprobes, and were able to successfully monitor parallel amplification of both amplicons and confirm their synthesis using subsequent melt curve analysis.

Having demonstrated the ability of the Eprobe technology, Riken and DNAForm are now exploring ways to apply the probes to various research and diagnostic assays.

Although Riken and DNAForm collaborated to develop Eprobes, DNAForm applied for patents surrounding the technology and has a license to the IP. "Currently, DNAForm designs and supplies Eprobes based on each requirement from customers," Usui said. "In the near future, DNAForm aims to commercialize [for] research purposes [an] Eprobe PCR kit for SNP genotyping, et cetera. Furthermore, the company has a motivation to develop molecular diagnostic kits."

Yasumasa Kimura, acting general manager of the molecular diagnosis division at DNAForm, confirmed in an email to PCR Insider that Eprobes "can be used for both molecular biology research and molecular diagnostics," and that the company is currently "negotiating with some other commercial entities [for] further development and commercialization."

Adding to their allure, "Eprobes can be used [with] any PCR primer and real-time PCR instrument," Usui said. "The emission/fluorescence combination of Eprobe is [the] same as SYBR Green I, and [uses the same] fluorescence filter without further modifications. Additionally, to [easily and rapidly] design adequate Eprobes for each PCR-amplified target, we're also trying to develop user-friendly Eprobe-designing software."

Another step the researchers are considering is boosting the multiplexing power of the probes. Although they demonstrated in their paper the discrimination of probes with two different fluorescent dyes, "combining multiple Eprobes with different dyes and melting temperatures could become an attractive approach for the design of highly complex superplexed PCR assays," the researchers wrote.

And although additional dyes have previously been described for ECHO probes, they may not be ideal for use with the Eprobe technology. For instance, one potential dye was found to not be thermostable, making it useless in PCR amplification.

"Therefore, additional thermostable dyes for Eprobes are desirable to extend the multiplexed abilities of Eprobes in the future," the researchers wrote.

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