- 1:55 PM
65e

Effect of Serum Contaminants on the Detection of DNA and RNA Using Surfactant Probes in Capillary Electrophoresis

Oxana Selivanova and James W. Schneider. Chemical Engineering, Carnegie Mellon University, 700 Technology Drive, Room 4307, Pittsburgh, PA 15219

Quantifying DNA and RNA sequences is a key process in the analysis of gene expression and identification of microorganisms. Gene expression quantification such as microarray methods is necessary for tracking the levels of messenger RNA (mRNA) in cells. mRNA purification from cells requires multiple steps due to different cellular constituents such as proteins and lipids present. Significant savings in cost and time could be realized if levels of DNA and RNA are measured in minimally processed samples, which would contain significant amounts of proteins, lipids, and other contaminants.

Micellar Electrokinetic Chromatography (MEKC) employs a surfactant probe that transiently interacts with micelles via its hydrophobic tail. Uncharged micelles migrate with electroosmotic flow and speed up the mobility of the probe. The resultant mobility of the probe is the average between its free solution mobility and micelle mobility. The probe sequence specifically hybridizes to complementary DNA/RNA shifting its mobility thus allowing quantification of the desired nucleic acid. Cellular contamination such as serum proteins can adsorb to the walls of bare silica capillaries thus requiring employment of coated capillaries. Proteins also result in micelle swelling. We are interested in the maximum amount of serum proteins that can be used with bare silica capillary for the successful DNA/RNA quantification. Alkylated DNA (aDNA) surfactant probe sequence specifically hybridizes to single stranded (ss) DNA creating aDNA/DNA duplex which partitions into MEKC micelles. The resultant output is an electropherogram where DNA is quantified via duplex mobility shift and peak intensity. Proteins are known to interact with DNA via G-C bases and DNA phosphodiester backbone. These interactions lead to the emergence of multiple and distorted peaks. Our results suggest that the probe prevents DNA from protein interaction. The position of overhanging DNA bases is crucial because hindered by the hydrophobic tail bases show no protein-DNA interaction. Only two peaks appear corresponding to aDNA and aDNA/DNA duplex as judged by a control without goat serum. Goat serum under 1mg/mL has no effect on DNA detection. At 2mg/mL significant peak distortion possibly due to protein adsorption onto bare silica capillary occurs. The next step is to detect longer ssDNA which is representative of cellular mRNA.