title: 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. ssDNA 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 poly(N-hydroxyethylacrylamide)-coated microchannel walls led to appreciable band-broadening, while much sharper peaks were seen for bioconjugates with nearly charge-neutral protein drag-tags.
authors.affiliation: Department of Chemical Engineering, Stanford University, Stanford, CA 94305-5444, USA.
authors.forename: Jennifer Coyne
authors.initials: JC
authors.lastname: Albrecht
authors.forename: Matthew B
authors.initials: MB
authors.lastname: Kerby
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Niedringhaus
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Lin
authors.forename: Xiaoxiao
authors.initials: X
authors.lastname: Wang
authors.forename: Annelise E
authors.initials: AE
authors.lastname: Barron
mesh_terms: D018736:DNA Adducts / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D046209:Electrophoresis, Microchip / Q000379:methods*; D005898:Glass / Q000737:chemistry; D015336:Molecular Probe Techniques; D015335:Molecular Probes / Q000737:chemistry* / Q000378:metabolism; D015203:Reproducibility of Results
chemical_list: D018736:DNA Adducts; D004277:DNA, Single-Stranded; D015335:Molecular Probes