Nonlinear electrophoretic response yields a unique parameter for separation of biomolecules

Abstract

We demonstrate a unique parameter for biomolecule separation that results from the nonlinear response of long, charged polymers to electrophoretic fields and apply it to extraction and concentration of nucleic acids from samples that perform poorly under conventional methods. Our method is based on superposition of synchronous, time-varying electrophoretic fields, which can generate net drift of charged molecules even when the time-averaged molecule displacement generated by each field individually is zero. Such drift can only occur for molecules, such as DNA, whose motive response to electrophoretic fields is nonlinear. Consequently, we are able to concentrate DNA while rejecting high concentrations of contaminants. We demonstrate one application of this method by extracting DNA from challenging samples originating in the Athabasca oil sands.

Conflict of interest statement

Conflict of interest statement: J.P., D.B., H.-L.P., and A.M. have a financial interest in Boreal Genomics, exclusive licensee of the technology presented in this paper.

Figures

Fig. 1.

SCODA concentration sequence. Time-lapse sequence showing concentration of SYBR Green I–stained pUC19 DNA (2.7 kb) from a homogeneous solution of 0.2 ng/μL of DNA in 1% agarose and 0.25× TBE, to a 750-μm-diameter spot. Images are taken at 10-min intervals, for a total run time of 60 min at a SCODA field of 250 V/cm (maximum field in SCODA gel). The concentration of DNA in the focused spot is estimated to be 100–200 ng/μL. (A) Diagram of dipole and quadrupole SCODA field lines. (B–H) SCODA duration, in minutes: B = 0, C = 10, D = 20, E = 30, F = 40, G = 50, and H = 60. Camera exposure is reduced to avoid saturation from increasing fluorescence intensity over the course of concentration. Exposure times, in milliseconds: B = 1,000, C = 750, D = 250, E = 50, F = 50, G = 10, and H = 10.

Fig. 2.

SCODA gel and injection chamber. SCODA gel boat for electrokinetic injection and concentration shown with a 60-μg/mL humic acid sample in the injection chamber. As indicated in the overlay, electrodes placed at locations 1 and 3 allow for application of a DC electric field to inject negative ions into the gel, where the rotating SCODA fields (applied at electrodes 1′, 2, 3, and 4) concentrate and trap molecules with a high ratio of k/D, whereas low-k/D molecules do not concentrate. This allows selective trapping and concentration of nucleic acids in the center of the gel. 1′ is a high-impedance electrode only used to monitor and clamp potentials; current is sourced at 1. Opposite electrodes (1′ and 3, 2 and 4) surrounding the SCODA gel are spaced 25 mm apart.

Fig. 3.

Injection and concentration sequence. Time-lapse sequence demonstrating injection and concentration of 200 ng of SYBR Green 1–stained pUC19 DNA. DNA is injected from 5 mL of 0.05× TBE buffer into a 1% agarose gel made with 0.25× TBE buffer. (A) Image taken after 10 min of injection at 20 V/cm. (B) Image taken after 10 min of subsequent SCODA with a maximum field of 250 V/cm. (C and D) Images taken at incremental 20-min SCODA concentration intervals for a total run time of 60 min. Camera exposure, in milliseconds: A = 1,000, B = 500, C = 100, and D = 20.

Fig. 4.

Electrophoretic washing. Time-lapse sequence showing dispersion and removal of contaminants concurrently with concentration of the desired nucleic acids. Images are a continuation of the experiment shown in Fig. 2, where 200 ng of pUC19 DNA was spiked into a 60-μg/mL humic acid solution, injected, and concentrated with superimposed electrophoretic washing. The increasing clarity of the gel indicates that the negatively charged humic acid contaminants are migrating out of the gel under the applied fields. (A–C) Visible light images showing decreasing humic acid (brown stain) contamination of the gel: in A after 20 min of DC injection, in B after an additional 10 min of SCODA concentration with DC field applied to wash contaminants from the gel, and in C after an additional 50 min of concentration with DC field. (D) UV-transilluminated image (100-ms exposure) taken at the same time point as C (80 min total elapsed time), in which stained DNA is clearly visible in the center of the gel.

Fig. 5.

Humic acid contaminant rejection. Success of PCR amplification of 2-ng samples of ABI Quantifiler Human DNA Standard containing increasing humic acid mass (horizontal axis, logarithmic scale), after extraction with silica column, magnetic bead, and SCODA purification methods. The 50% cutoff for SCODA is beyond the range of humic acid concentrations that could be mixed from commercially available humic acid stocks. Projected performance of SCODA in humic acid rejection is at least 100-fold better than the next commercially available method that is not specifically designed to reject humic acids.

Fig. 6.

Oil sands. Left: raw oil sand sample, showing bitumen, water, sand, and clay mixture. Right: Resuspension of oil sands mixture in buffer for DNA extraction from SCODA.

Fig. 7.

Conservation of oil sand–associated environmental DNA. Vector and quality trimmed fosmid sequences prepared from SCODA-concentrated DNA were aligned to GENBANK-nt using wuBLAST. The percentage sequence identity distribution for high-scoring pairs (HSPs) follows a Gaussian distribution. The bulk of the fosmid sequences have restricted sequence identity (≈30–70%) compared with known genomes. A partial legend for best scoring hits is shown; for a complete list, see Table S2.