New Dual-Functional Fluorescent Probe for DNA Hybridization and Melting Point Analysis

Japanese researchers have described a new approach for combining real-time PCR amplification and melting curve analysis in a single-tube reaction, which provides a powerful means for new mutation detection assays.

This technology, which was jointly developed by the RIKEN Center for Life Science Technologies (Saitama, Japan) and K.K. DNAFORM (Yokohama City, Japan) will enable the development of new, advanced assays for DNA-based genetic testing and should help to bring the benefits of genome-wide sequencing studies to patients in the clinic.

The new methodology is based on RIKEN's Eprobe, a highly efficient and reliable fluorescent probe. Eprobes contain two dye moieties attached to the same nucleotide, and their fluorescent signal is strongly suppressed as single-stranded oligonucleotides by an excitonic interaction between the dyes. Upon hybridization to a complementary DNA strand, the dyes are separated and intercalate into the double-strand leading to strong fluorescence signals. Furthermore, intercalation of dyes further stabilizes the DNA/DNA hybrid and increases the melting temperature compared to standard DNA oligonucleotides. Thus, Eprobes allow for specific real-time monitoring by amplification while enabling study of reaction products by melting curve analysis.

In a recent study, the investigators used Eprobes for the detection of genetic variations in a human EGFR (epidermal growth factor receptor) and KRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) tumor. They demonstrated that Eprobes provided decisive advantages over commonly used hybridization probes because of their unique background signal reduction, enhanced DNA-binding affinity, and very low false positives rate.

"We are very excited about the potential of the new Eprobes,” said Dr. Matthias Harbers, a visiting scientist at the RIKEN Center for Life Science. "In the reactions, Eprobes acted like sequence-specific fluorescent dyes, which gives them great potential for use as hybridization probes not only in PCR and melting curve analysis but also in other important applications like, for instance, in fluorescent in situ hybridization or FISH."

The Eprobe study was published in the August 7, 2013, online edition of the journal PLOS ONE.

Related Links:
RIKEN Center for Life Science Technologies
K.K. DNAFORM