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.concentration ͉ DNA ͉ electrophoresis ͉ purification ͉ SCODA M ethods for separating different molecular species are the cornerstone of analytic techniques in molecular biology. Of these, nucleic acid extraction from complex sources is a molecular separation problem of great importance to current challenges in genomics, metagenomics, forensics, biodefense, food and water safety, and clinical molecular diagnostics.The ubiquitous column-and bead-based nucleic acid extraction methods that dominate the field of nucleic acid extraction use selective chemical affinity between nucleic acids and ion exchange or similar resins and beads to capture target molecules. Although these methods often involve mechanical steps including filtration and centrifugation, they are relatively inexpensive and work well in a variety of samples. Their inadequacy lies in the fact that the separations are based on chemical affinity and therefore perform poorly in the presence of contaminant molecules that either have similar chemical properties to nucleic acids or foul the capture matrix (1). Precipitation methods are often used after column or bead extractions to remove contaminants that carry through; however, this further reduces yield of the methods, particularly in cases with low target concentrations.This weakness of existing methods is a critical problem for DNA extraction from environmental samples. For example, humic acids, a family of contaminants abundant in soil, coextract with DNA in phenol-based separations owing to their solubility in the aqueous phase (1) and partly carry through column-and bead-based methods. The situation worsens with a low starting concentration of nucleic acids because the dual challenge must then be faced of concentrating few nucleic acids while rejecting large amounts of contaminants. A universal, simple, and highly selective method to concentrate DNA from contaminated and low-abundance sources would be very desirable.We have found a unique solution to this problem in the physics of electrophoresis. It has long been known that nucleic acid molecules, because of their exceptionally long contour lengths and high linear charge density, exhibit complex electrophoretic...

Efficient Genomic DNA Extraction from Low Target Concentration Bacterial Cultures Using SCODA DNA Extraction Technology

“…It should also be noted that SCODA is fundamentally different from other electrophoretic separation www.cshprotocols.org 3 Cold Spring Harbor Protocols and sample preparation techniques (such as PFGE) because SCODA can concentrate all DNA molecules above ~300 bp into a buffer sample while simultaneously removing contaminants. Other electrophoretic mechanisms are unable to concentrate samples, and also do not provide a method for effective contaminant removal (Pel et al 2009). This protocol highlights SCODA's strengths as a general method for recovering small amounts of DNA from contaminated samples (Broemeling et al 2008;Pel et al 2009).…”

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