Resolution of multiple ssDNA structures in free solution electrophoresis

By using high concentrations of buffer, electroosmotic flow within uncoated channels of a microfluidic chip was minimized, allowing the free solution electrophoretic separation of DNA. More importantly, because of the ability to efficiently dissipate heat within these channels, field strengths as high as 600 V/cm could be applied with minimal Joule heating (<2 degreesC). As a result of the higher field strengths, separations within an 8-cm-long channel were achieved within a few minutes. However, when the electrophoretic separation of single-stranded DNA (ssDNA) less than 22 bases in length was performed, containing the fluorophore Texas Red as an end label, more than the expected single peak was observed at this high electric field. On the other hand, the free solution electrophoresis of a double-stranded DNA (dsDNA) consisting of a random sequence did exhibit the expected single peak. The appearance of these multiple peaks for ssDNA is shown to be dependent upon the base content and sequence of the ssDNA as well as on the chemical structure of the fluorophore used to tag the DNA for detection. Specifically, the peaks can be attributed to different secondary structures that result either from hydrophobic interactions between the DNA bases and an uncharged fluorescent dye or from G-quadruplexes within guanine-rich strands.