Fluorescent and electrochemical labels help scientists detect genetic disorders that can cause cancer
A team of Japanese scientists led by Akimitsu Okamoto from the RIKEN Frontier Research System, Wako, has developed a new method for tagging a particular DNA base responsible for causing cancer.
Cytosine, a common DNA base, is reacted to add a methyl group to form methylcytosine during many biological processes. This process, known as methylation, is important for gene regulation, and DNA and protein stability. Further, excessive methylation of cytosine has been shown to result in cancer. The development of simple techniques to detect methylcytosine is therefore of great interest to scientists.
Although conventional methods have many advantages, they also have problems. Current methods cannot differentiate between cytosine and methylcytosine; they also destroy the DNA sample and are time-consuming. The latest technique by Okamoto and co-workers is selective for methylcytosine, fast and allows easy detection1.
The technique takes advantage of the easy oxidation of methylcytosine and uses three, specially designed, components to enable detection. When the reaction takes place, the methylcytosine forms a stable complex with an oxidant, potassium osmate, and a rate-enhancing ligand. The ligand, a bipyridine derivative, can then react further to bond with a variety of fluorescent or electrochemical tags allowing routine detection of the complex (Fig. 1 - Click on link below).
This conceptually new approach to methylcytosine detection takes just six hours to complete. Importantly, the key complex only forms between the methylcytosine and the ligand. This leaves the cytosine in the sample untouched and allows a clear distinction to be made. In addition, methylcytosines in single-stranded DNA efficiently formed the complex, whereas complexation of methylcytosines in a DNA duplex was suppressed. This result implies that the technique could also provide sequence-specific results giving detailed and accurate information of the methylated sites.
Okamoto explains that there is still more work to be done. Unfortunately, the information gained from the sequence-specific studies is limited as a consequence of the competing reaction with thymine, another DNA base. Also, the signal intensities and sensitivities are a little too weak to be useful on small sample sizes at this time.
Okamoto and his team are now striving to improve their technique so it can be used routinely in clinics with standard fluorescence or electronic signal analyzers. This technique is based on easy-to-use chemistry and Okamoto says, “Because the total process finishes in a few hours, this technique may make it possible to design machines that automate a series of processes from purification of samples to analysis.”
Reference
1. Tanaka K., Tainaka K., Kamei T. & Okamoto A. Direct labeling of 5-methylcytosine and its applications. Journal of the American Chemical Society 129, 5612–5620 (2007).
For more information, please contact
Saeko Okada
Email: [email protected]
