Eprobe®, which was jointly developed by DNAFORM and RIKEN, is a synthetic oligonucleotide-based fluorescent probe that has an excitonic interaction. Performing polymerase chain reaction (PCR) with Eprobe® allows for specific quantification of target DNA sequences and also enables base mutation detection.

□Function of Eprobe® in real-time PCR

Eprobe® is a fluorescence-labeled oligonucleotide with one modified thymine carrying two dye moieties. When Eprobe® forms a double-stranded structure with target DNA sequences, the two dyes in Eprobe® are separated and emit a strong fluorescence caused by disruption of the excitonic interaction between dyes. Eprobe®, when added in sufficient amounts to the reaction mixture for real-time PCR, also binds to its target DNA sequence during the primer annealing step. By measuring the fluorescence generated upon binding of Eprobe®, only the target DNA sequence is quantified.

This Eprobe®-mediated real-time PCR can also be applied to the detection of base mutations. In post-PCR melting curve analysis, differences in melting temperature (Tm) between when Eprobe® binds to a perfectly matched wild-type base sequence and when the same Eprobe® binds to mismatched mutated base sequence(s) are expected. By analyzing the differences in Tm, the presence or absence of mutated DNA sequence(s) can be assessed.

□Features of Eprobe® PCR

• □Eprobe® generates a fluorescence signal upon hybridization to its target nucleotide sequence.
• □The use of a single Eprobe® allows for both real-time PCR and melting curve analysis.
• □Higher melting temperature (Tm values) compared to normal DNA oligonucleotides are observed.

□Applications

• □Highly sensitive detection of bacteria and viruses.
• □Quantification of gene expression.
• □Detection of SNPs.
• □Detection of somatic mutations in disease-causing genes such as cancer.

For more information on Eprobe® PCR, please see the Eprobe®/Eprimer™ Technical Information.

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