Method for determination and quantification of radiation or genotoxin exposure
The present invention discloses methods for detecting exposure of a living subject to genotoxic agents, testing sensitivity to a genotoxic agent, and determining DNA damage caused by exposure to an agent, comprising detecting the presence of FANCD2-containing foci from a sample collected from said subject. The presence of concentrated foci is indicative of DNA damage, and the degree of foci formation is correlated with degree of exposure. Diagnostic reagents contain a ligand that binds to human FANCD2 associated with a detectable label. Kits for detecting DNA damage in a biological sample contain such diagnostic reagents and signal detection components. The invention further discloses methods for identifying agents which modulate the ability of FANCD2-containing foci to form. Among other things, such agents are potentially useful chemosensitizing agents or may confer protection against damage caused by genotoxic agents.
1. A method of screening test agents which modulate formation of FANCD2-containing foci, said method comprising:
identifying an individual suspected of having been exposed to a genotoxic agent;
obtaining a biological sample from said individual;
contacting said biological sample with test compound; and
detecting the presence of FANCD2 in FANCD2-containing foci relative to a control sample, wherein a difference in the degree of foci formation relative to a control sample is indicative of an agent active in foci formation, wherein said biological sample is a cell expressing a polynucleotide comprising a DNA sequence encoding the human FACD2 protein of SEQ ID NO:1 fused in frame with a DNA sequence encoding a fluorescent protein.
2. The method of claim 1 , wherein said detecting comprises fluorescence microscopy.
3. The method of claim 1 , wherein said detecting comprises measuring fluorescence resonance energy transfer.
4. A method of screening test agents which modulate formation of FANCD2-containing foci, said method comprising:
identifying an individual suspected of having been exposed to a genotoxic agent;
obtaining a biological sample from said individual;
contacting said biological sample with test compound; and
detecting the presence of FANCD2 in FANCD2-containing foci relative to a control sample, wherein a difference in the degree of foci formation relative to a control sample is indicative of an agent active in foci formation, wherein said biological sample is a cell expressing a polynucleotide comprising a DNA sequence encoding a protein that binds with FANCD2 upon foci formation, fused in frame with a DNA sequence encoding a second fluorophore.
5. A method of screening test agents which modulate formation of FANCD2-containing foci, said method comprising:
identifying an individual suspected of having been exposed to a genotoxic agent;
obtaining a biological sample from said individual;
contacting said biological sample with test compound; and
detecting the presence of FANCD2 in FANCD2-containing foci relative to a control sample, wherein a difference in the degree of foci formation relative to a control sample is indicative of an agent active in foci formation, wherein said biological sample is a cell expressing a polynucleotide comprising a DNA sequence encoding the human FACD2 protein of SEQ ID NO:1 fused in frame with a DNA sequence encoding a fluorescent protein and a polynucleotide comprising a DNA sequence encoding a protein that binds with FANCD2 upon foci formation, fused in frame with a DNA sequence encoding a second fluorophore.