Free‐solution electrophoretic separations of DNA–drag‐tag conjugates on glass microchips with no polymer network and no loss of resolution at increased electric field strength

Abstract

Here, we demonstrate the potential for high-resolution electrophoretic separations of ssDNA-protein conjugates in borosilicate glass microfluidic chips, with no sieving media and excellent repeatability. Using polynucleotides of two different lengths conjugated to moderately cationic protein polymer drag-tags, we measured separation efficiency as a function of applied electric field. In excellent agreement with prior theoretical predictions of Slater et al., resolution is found to remain constant as applied field is increased up to 700 V/cm, the highest field we were able to apply. This remarkable result illustrates the fundamentally different physical limitations of Free-Solution Conjugate Electrophoresis (FSCE)-based DNA separations relative to matrix-based DNA electrophoresis. Single-stranded DNA separations in “gels” have always shown rapidly declining resolution as the field strength is increased; this is especially true for ssDNA > 400 bases in length. FSCE’s ability to decouple DNA peak resolution from applied electric field suggests the future possibility of ultra-rapid FSCE sequencing on chips. We investigated sources of peak broadening for FSCE separations on borosilicate glass microchips, using six different protein polymer drag-tags. For drag-tags with four or more positive charges, electrostatic and adsorptive interactions with pHEA-coated microchannel walls led to appreciable band-broadening, while much sharper peaks were seen for bioconjugates with nearly charge-neutral protein drag-tags.

Simultaneous detection of 19 K‐ras mutations by free‐solution conjugate electrophoresis of ligase detection reaction products on glass microchips

“…As mentioned previously, FSCE theory predicts that no loss of resolution is expected as electric field strength is increased . Theory predicts that as the field strength increases, peak width decreases faster than the peak spacing.…”

“…Microchip electrophoresis separations were performed on a home‐built electrophoresis system that was previously described . A 488 nm solid‐state laser was used to excite the fluorescent dye‐labeled DNA, and the emitted light passed through a dichroic filter before being detected on a cooled CCD.…”

“…Multiplexed separations of these 19 mutations were performed using both capillary and microchip electrophoresis in free solution with no polymer network. Additionally, microchip separations highlighted an advantage of FSCE over polymer matrix‐based separations; no loss of resolution occurred when the electric field strength was doubled . The 19 LDR peaks were separated in less than 75 s on a glass microchip at the highest field strength possible.…”

Microfluidic electrophoretic mobility shift assays for quantitative biochemical analysis

“…A drag‐tag‐induced mobility shift has been shown to improve the separation resolution, especially for samples that are otherwise difficult to fractionate. The Barron group developed microfluidic “drag‐tag” electrophoretic tools for a series of applications including detection of SNPs , detection of point mutations with ligase detection reaction , determination of the primer‐dimer formation risk , and DNA size‐based separations in free solution. For the primer‐dimer formation , the “drag‐tag” is used to enhance the separation resolution in the free solution between the single‐stranded primer and the double‐stranded dimer that are of identical mass/charge ratio.…”

“…binding affinity, melting point). In a DNA sizing assay , a positively charged protein polymer was conjugated to the target oligonucleotide and subsequent free‐solution electrophoretic separation reported molecular size. The protein‐based drag‐tag significantly changed the size‐to‐charge ratio, amplifying the mobility difference between oligonucleotides with only a few base pair differences.…”