Analysis of bacterial plasmid DNA by solid-phase microextraction

Abstract

The extraction and preconcentration of DNA is a critical step in the analysis of microorganisms. In this study, a polymeric ionic liquid (PIL) sorbent coating was applied for the preconcentration of plasmid DNA (pDNA) from bacterial cells using solid-phase microextraction (SPME). PIL-based SPME devices were prepared by ultraviolet photoinitiated polymerization of a dicationic ionic liquid (IL)-based cross-linker and IL monomer on a nitinol support. pDNA was extracted from buffered aqueous solution using the PIL-based sorbent coating followed by the amplification of a target gene by polymerase chain reaction (PCR). Extraction conditions for the method were optimized based on the relative intensities of PCR amplicon bands visualized on an agarose gel. Compared to a commercial polyacrylate sorbent coating, the PIL sorbent coating extracted greater quantities of pDNA. With an extraction time of 5 min, the PIL-based SPME technique was capable of preconcentrating a sufficient amount of template pDNA from a 20 ng mL⁻¹ solution to allow detection of the amplicon on an agarose gel. Sequence analysis demonstrated that the sequence of the pDNA was unaltered following PIL-based SPME. The developed method was successfully employed for the analysis of pDNA from two different E. coli transformants in a dilute aqueous solution.