Novel electrophoresis mechanism based on synchronous alternating drag perturbation

Marziali, Andre; Pel, Joel; Bizzotto, Dan; Whitehead, Lorne

doi:10.1002/elps.200406140

Cited by 32 publications

(40 citation statements)

References 19 publications

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“…1H). The radial asymmetry in the pattern formed during the first minutes of focusing results from the fact that the fields applied to the gel are discrete approximations (both in spatial geometry and in time) of the ideal dipole and quadrupole fields (4).…”

Section: Resultsmentioning

confidence: 99%

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Nonlinear electrophoretic response yields a unique parameter for separation of biomolecules

Pel

Broemeling

Mai

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Resultsmentioning

confidence: 99%

“…1), in a method termed synchronous coefficient of drag alteration (SCODA) (4,5), for generating a divergent velocity field that is capable of selectively concentrating nucleic acids in a gel. A brief explanation of the method follows.…”

mentioning

confidence: 99%

Nonlinear electrophoretic response yields a unique parameter for separation of biomolecules

Pel

Broemeling

Mai

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…However, the extraction and purification of nucleic acids in feces is quite challenging due to its low abundance and the high level of contaminants like humic acid. Thankfully, there are a number of commercial kits available for fecal DNA isolation, and new techniques such as synchronous coefficient of drag alteration (SCODA) show promise in further purifying and concentrating this rare DNA (Broemeling et al, 2008;Marziali et al, 2005). Interestingly, both the amount and integrity of DNA in feces have been shown to identify colorectal cancer patients (Klaassen et al, 2003;Osborn & Ahlquist, 2005).…”

Section: Fecesmentioning

confidence: 99%

Novel Tissue Types for the Development of Genomic Biomarkers

Sergueeva¹,

Collins²,

Dow³

et al. 2012

Biomarker

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“…Remarkably, a relatively larger device (on the order of a few cm) filled with bulk agarose gel has been demonstrated for the purification and concentration of high molecular weight DNA (on the order of tens of thousands of bp), using the field-dependent mobility of DNA in a method called synchronous coefficient of drag alteration (SCODA) [17][18][19] . However, this study overlooked the fact that the field-dependent mobility depends on the DNA fragment size, which will be shown for the first time in this work to provide an alternative method for continuous flow DNA fractionation that has hitherto not been demonstrated.…”

Section: Introductionmentioning

confidence: 99%

Exploiting biased reptation for continuous flow preparative DNA fractionation in a versatile microfluidic platform

et al. 2017

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A new approach is presented for preparative, continuous flow fractionation of sub-10-kbp DNA fragments, which exploits the variation in the field-dependent mobility of the DNA molecules based on their length. Orthogonally pulsed electric fields of significantly different magnitudes are applied to a microchip filled with a sieving matrix of 1.2% agarose gel. Using this method, we demonstrate a high-resolution separation of 0.5, 1, 2, 5, and 10 kbp DNA fragments within 2 min. During the separation, DNA fragments are also purified from other ionic species. Preparative fractionation of sub-10-kbp DNA molecules plays an important role in second-generation sequencing. The presented device performs rapid high-resolution fractionation and it can be reliably manufactured with simple microfabrication procedures. This method has the advantages of simplicity, versatility, and reproducibility. However, it suffers from long processing times on the order of a few tens of hours 2,3 . For instance, the Bio-Rad CHEF-DR device performs fractionation of 5-120 kbp DNA using pulsed-field gel electrophoresis (PFGE) in 25 h (Refs. 4,5). Trends toward second-generation sequencing motivate the replacement of standard gel electrophoresis with microchip-based systems, which could provide efficient platforms to minimize the processing time and to perform optimal DNA fractionation 6-8 .Micro-and nanofabricated post arrays-that resemble a gel matrix with well-ordered and identical pores-have been integrated in microchip-based systems, increasing both the understanding of DNA separation principles and the fractionation's efficiency and speed. Two decades ago, Volkmuth and Austin 9 introduced a patterned micro-post array in a microfluidic electrophoresis platform, in which DNA fragments were separated by biased reptation under a DC electric field. Later, Duke et al. separated large fragments in a range of 60-135 kbp in a microfabricated array using DNA reorientation-or the 'switchback' principle. Although the switchback principle is similar to what is used in PFGE, the device yields much faster separations owing to its sparse and regular sieving array 10 . In a later work, Kaji et al. studied a three-dimensional (3D) nanopost array as an optimal separation matrix for DNA fragments over a few kbps under a DC electric field, resulting in similarly fast separations due to the sparse matrix 11 .With the aim of increasing the sample throughput and further facilitating the sample recovery, a continuous flow separation was developed. Huang et al. 12 performed continuous flow PFGE in a micromachined post array by applying pulsed electric fields of slightly unequal strengths. DNA fragments ranging between 61 and 209 kbp were separated within 15 s in the 'DNA prism'.

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Novel electrophoresis mechanism based on synchronous alternating drag perturbation

Cited by 32 publications

References 19 publications

Nonlinear electrophoretic response yields a unique parameter for separation of biomolecules

Nonlinear electrophoretic response yields a unique parameter for separation of biomolecules

Novel Tissue Types for the Development of Genomic Biomarkers

Exploiting biased reptation for continuous flow preparative DNA fractionation in a versatile microfluidic platform

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