Fluorescence Polarization Combined Capillary Electrophoresis Immunoassay for the Sensitive Detection of Genomic DNA Methylation

Abstract

Genomic DNA hypomethylation is epigenetically associated with aberrant gene expression and chromosome instability. Here we describe a method for rapid and sensitive detection of genomic DNA methylation without the need for bisulfite conversion, enzymatic digestion, or PCR amplification. The methylated DNA is first specifically recognized by an anti-5-methylcytosine IgG1 antibody and noncovalently labeled by a monovalent, fluorescently labeled, Fc-specific anti-IgG1 Fab fragment (secondary antibody). The formed immuno-complex of methylated DNA can be efficiently focused and separated from the DNA unbound secondary antibody by capillary electrophoresis (CE). The free fluorescent dye comigrates with the immuno-complex. However, by taking advantage of online laser-induced fluorescence polarization detection (LIFP), the target immuno-complex can be distinguished and accurately measured from the overlapped dye without further separation. The developed method is highly sensitive with a LOD of 0.3 nM and is highly specific for the detection of methylated DNA. Moreover, the CE-LIFP immunoassay is rapid (1.2 min for one analysis) and only consumes less than 0.1 ng of genomic DNA. The method was validated by examining human cell lines treated by methyltransferase inhibitor 5-aza-2'-deoxycytidine at low doses (10 nM-10 microM). Because of its sensitivity and speed, our method will be applicable for rapid epigenetic evaluation and for the study of tumorigenesis and chemical-cell interactions.