Abstract 1704: AC electrokinetic isolation of cell free high molecular weight DNA (CF-HMW DNA) from serum

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Recent reports of CF-HMW DNA in a variety of diseases has led to interest in its use as a prognostic biomarker, a diagnostic or screening tool for cancer. Apoptotic DNA ( 500bp) that is then trafficked into circulation. Dielectrophoresis, or DEP (movement of particles in a non-uniform AC electric field), based electrokinetic techniques have been used for protein biomarker screening, cell separation, and DNA manipulation. Using specific DEP frequencies, analytes are moved to known locations (high or low field region) based on their inherent dielectric properties. The process allows specific nano-sized particles to be highly concentrated into high field regions, while micron-size cells are held weakly in low field regions. With fluidic washing, cells can be removed while nano-sized particles are held in high field regions. This method can effectively act as a CF-HMW DNA enrichment process for biological fluids. Unfortunately, DEP does not work well in (high conductance) physiological buffers, requiring significant dilution of the sample and, as a result, the target analyte concentration. Here, we describe a novel AC electrokinetic (ACE) technique using a custom microelectrode array and AC parameters to isolate and analyze CF-HMW DNA from high conductance serum. DNA from normal serum samples and the A549 cell line were isolated using Qiagen kits to determine initial DNA quantities. Normal sera were spiked with A549 cell line DNA at concentrations from 0 to 500 ng/ml and subjected to a 20 minute ACE protocol. DNA isolation was visualized in situ using SYBR Green I fluorescence and then eluted for use in secondary analyses. Results showed minimal fluorescence from samples without spike whereas A549 DNA spiked samples showed concentration-dependent fluorescence on the microelectrodes. After elution, levels of DNA were quantitated using ALU-sequence based qPCR and K-ras mutational status was confirmed by ABI CastPCR. Future experiments will compare CF-HMW DNA levels from normal and cancer patients across a variety of solid tumors. The ACE technique enables rapid isolation and detection of CF-HMW DNA directly from biological fluids without prior sample prep and has broad utility for diagnosis and monitoring of cancer and other diseases in which CF-HMW DNA may be used as a marker. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1704. doi:1538-7445.AM2012-1704