Fluorescently Imaged Particle Counting Immunoassay for Sensitive Detection of DNA Modifications

摘要

Modifications of genomic DNA may change gene expressionnand cause adverse health effects. Here we for the firstntime demonstrate a particle counting immunoassay fornrapid and sensitive detection of DNA modifications usingnbenzo[a]pyrenediol epoxide (BPDE)-DNA adducts as annexample. The BPDE-adducted DNA is specifically capturednby immunomagnetic particles and then isolatednfrom unmodified DNA by applying an external magneticnfield. By taking advantage of the fluorescence signalnamplification through multiple labeling of captured DNAnby OliGreen dye, the captured BPDE-DNA adducts cannbe quantified by particle counting from fluorescencenimaging. This clearly demonstrates that the number ofnfluorescently countable particles is proportional to thenmodification content in genomic DNA. It is interesting tonnote that the background fluorescence signal caused bynnonspecific adsorption of OliGreen dye can be moreneffectively quenched than that induced by the binding ofnOliGreen dye to ssDNA, allowing for significant reductionnin the background fluorescence and further enhancing thendetection sensitivity. The developed method can detectntrace BPDE-DNA adducts as low as 180 fM in thenpresence of 1 billion times more normal nucleotides inngenomic DNA and has a dynamic range over 4 orders ofnmagnitude. By using anti-5-methylcytosine antibody, thenmethod is extended to the detection of global DNAnmethylation. With high sensitivity and specificity, thisnrapid and easy-to-perform analytical method for DNAnmodifications shows a broad spectrum of potential applicationsnin genotoxical and epigenetic analysis.