(63d) Purification of Genomic DNA from Whole Cell Lysates Using Photoactivated Polycarbonate Microfluidic Devices

The ability to analyze cells mutational content via signature sequences elucidated from their genomic DNA (gDNA) requires the ability to recover and purify the gDNA from the whole cell lysate. Following cell lysis, it is often necessary to remove cellular debris/proteins that interfere with the subsequent bioenzymatic reactions. We will discuss the use of a photoactivated polycarbonate (PPC) microfluidic device for the surface-immobilization and purification of gDNA from whole cell lysates. The immobilization bed for gDNA consisted of an enclosed microchannel with 10 µm diameter, high aspect ratio microposts, which were fabricated during embossing of the microfluidic channels. The surface of the PC device was activated by ultraviolet radiation and a photooxidation reaction caused by UV light resulted in the production of primarily carboxylate groups. The gDNA was captured on this photoactivated surface in an immobilization buffer (IB), which contained 3% PEG, 0.4 M NaCl, and 70% ethanol. The process of immobilization and purification consisted of three steps: (i) suspension of crude cell lysates in the IB and insertion into the PPC device causing surface-immobilization of the gDNA, (ii) washing with 85% ethanol, and (iii) release from the surface with DI water. The assay was performed within 25 min. The presence of the purified gDNA was confirmed by the PCR with complementary primers to amplify a recognition sequence within the gDNA from the lysed cells. A load of approximately 8 µg/mL of gDNA was immobilized onto the PPC bed. The recovery of DNA following purification was estimated to be ~85%. Chip-to-chip reproducibility of the assay was ~15%. Results demonstrated the ability of the method to remove cellular proteins and debris from the cell lysate. The absorbance ratio of I260/I280 obtained after purification of whole cell lysates was found to be between 1.7-1.9 indicating that the method produced negligible protein contamination.