Fluorescent oligonucleotides can serve as suitable alternatives to radiolabeled oligonucleotides

Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA.
Journal of biomolecular techniques: JBT 10/2009; 20(4):190-4.
Source: PubMed


Prolonged exposure to radiation from radionuclei used in medical research can cause DNA damage and mutation, which lead to several diseases including cancer. Radioactivity-based experiments are expensive and associated with specialized training, dedication of instruments, approvals, and cleanup with potential hazardous waste. The objective of this study was to find an alternative to the use of radioactivity in medical research using nucleic acid chemistry. FITC-labeled oligonucleotides that contain wild-type (wt) and modified base (8-oxo-G) at the same position and their complementary unlabeled strand were synthesized. Purified DNA repair enzyme, OGG1, and nuclear lysates from MCF-7 breast cancer cells were incubated with double-stranded FITC-labeled wt and 8-oxo-G oligonucleotide to demonstrate the OGG1 incision assay. We found that FITC-coupled oligonucleotides do not impose a steric hindrance during duplex formation, and the fluorescence intensity of the oligonucleotide is comparable with the intensity of the radioactive oligonucleotide. Moreover, we have seen that the OGG1 incision assay can be performed using these fluorescence oligonucleotides, replacing conventional use of radiolabeled oligonucleotides in the assay. Although the use of fluorescent-labeled oligonucleotides was described in detail for incision assays, the technique can be applied to replace a broad range of experiments, where radioactive oligonucleotides are used, eliminating the hazardous consequences of radiation.

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Available from: Rahul Ballal, Jan 25, 2016
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    • "The enhanced ability to visualize and track biomolecules has formed the cornerstone of revolutionary growth in molecular biology and biochemistry (Massoud & Gambhir, 2003; Miyakawa, Sawano, & Kogure, 2003; Prescher & Bertozzi, 2005). Over the past couple of decades , advances in fluorescence technology have provided a more accessible route to tagging and imaging of nucleic acids compared with radioactivity (Ballal, Cheema, Ahmad, Rosen, & Saha, 2009). In this regard, the discovery of small molecules that form stable and highly fluorescent complexes with DNA has been a breakthrough in the development of DNA probes (Ahn, Costa, & Emanuel, 1996; Dragan et al., 2010; Glazer, Peck, & Mathies, 1990; Zipper, Brunner, Bernhagen, & Vitzthum, 2004). "
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    Full-text · Article · Jul 2013 · Journal of biomolecular Structure & Dynamics
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    • "The use of oligonucleotides with a fluorescent label has been shown to be a reliable alternative to radiolabelling [17]. For primer extension assays, primer oligonucleotide (P1) with a 5′ 6-FAM label was purchased from the Keck Centre (Yale University). "
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