Japanese investigators from the RIKEN Research Institute are reporting in the latest issue of the Journal of the American Chemical Society that they created fluorescent DNA probes that can attach to a specific DNA gene sequence, and detect single nucleotide alterations. The development of such PRODAN-conjugated DNA probes can pave the way for a diagnostic modality to identify single nucleotide polymorphisms (SNPs) responsible for a wide variety of genetic disorders:
SNP diagnostics have recently attracted much attention and several strategies to identify SNPs have been developed in the past few years. Existing methods are often limited by the need to identify large DNA sequences. Now, a team of Japanese researchers led by Akimitsu Okamoto from the RIKEN Frontier Research System, Wako, has used derivatives of the fluorescent dye PRODAN to correctly identify SNPs quickly and efficiently.
PRODAN, a well-known fluorophore, absorbs and emits light at different wavelengths depending on the polarity of its environment. Okamoto reasoned that similar dyes, which include differing nucleotide components, could be incorporated into DNA structures and ‘report back’ differences in the microenvironment. Such changes in the microenvironment would likely be the result of small changes in the DNA structure and allow detection of sequence variations. The team synthesized four variants of the dye, so that all combinations of base matches and mismatches could be investigated.
Once incorporated in a DNA sequence under a variety of conditions, the team calculated the differences between the wavelengths absorbed and emitted by the dye. These differences are known as Stokes shifts. The researchers detected a small Stokes shift when DNA base pairs matched correctly, but a larger shift when there was a mismatch. Therefore, by using various PRODAN-labeled DNA dyes, single nucleotide alterations could be detected. "The use of this DNA probe makes it possible to judge the type of base located at a specific site on the target DNA, simply by mixing the DNA and the dye together. This method is a very powerful assay that does not require enzymes or time-consuming steps, and avoids errors," says Okamoto.
Okamoto believes that this method of detection is very promising and is working towards making the system suitable for every day use. "I think SNP chips using our probe would make important contributions to cancer diagnosis," he enthuses.