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Fluorescent oligonucleotides can serve as suitable alternatives to radiolabeled oligonucleotides
- Source :
- Journal of biomolecular techniques : JBT. 20(4)
- Publication Year :
- 2009
-
Abstract
- 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.
- Subjects :
- Radioisotopes
DNA Repair
Recombinant Fusion Proteins
Ubiquitin-Protein Ligases
Genes, BRCA1
Deoxyguanosine
Nucleic Acid Hybridization
Breast Neoplasms
Articles
Adenocarcinoma
Transfection
DNA Glycosylases
Neoplasm Proteins
Substrate Specificity
Oligodeoxyribonucleotides
8-Hydroxy-2'-Deoxyguanosine
Cell Line, Tumor
Isotope Labeling
Humans
Female
Oligonucleotide Probes
Fluorescein-5-isothiocyanate
Fluorescent Dyes
Subjects
Details
- ISSN :
- 19434731
- Volume :
- 20
- Issue :
- 4
- Database :
- OpenAIRE
- Journal :
- Journal of biomolecular techniques : JBT
- Accession number :
- edsair.pmid..........1860dca7b0fa117ac3c313078bf1a3ee