High-Throughput Separation of Long DNA in Deterministic Lateral Displacement Arrays

Abstract

Length-based separation of DNA remains as relevant today as when gel electrophoresis was introduced almost 100 years ago. While new, long-read genomics technologies have revolutionised accessibility to powerful genomic data, the preparation of samples has not proceeded at the same pace, with sample preparation often constituting a considerable bottleneck, both in time and difficulty. Microfluidics holds great potential for automated, sample-to-answer analysis via the integration of preparatory and analytical steps, but for this to be fully realised, more versatile, powerful and integrable unit operations, such as separation, are essential. We demonstrate the displacement and separation of DNA with a throughput that is one to five orders of magnitude greater than other microfluidic techniques. Using a device with a small footprint (23 mm × 0.5 mm), and with feature sizes in the micrometre range, it is considerably easier to fabricate than parallelized nano-array-based approaches. We show the separation of 48.5 kbp and 166 kbp DNA strands achieving a significantly improved throughput of 760 ng/h, compared to previous work and the separation of low concentrations of 48.5 kbp DNA molecules from a massive background of sub 10 kbp fragments. We show that the extension of DNA molecules at high flow velocities, generally believed to make the length-based separation of long DNA difficult, does not place the ultimate limitation on our method. Instead, we explore the effects of polymer rotations and intermolecular interactions at extremely high DNA concentrations and postulate that these may have both negative and positive influences on the separation depending on the detailed experimental conditions.

Short and long-range cyclic patterns in flows of DNA solutions in microfluidic obstacle arrays

“…Recently, high throughput DNA sorting was demonstrated using DLD. 2 Throughputs reached 24 μL h −1 and 760 ng h −1 which corresponds to orders of magnitude improvement over existing microfluidic approaches. DLD has also been used for sorting of physiological concentrations of blood 3 at high throughputs.…”

“…Recently, high throughput DNA sorting was demonstrated using DLD 2 . Throughputs reached 24 µL/h and 760 ng/h which corresponds to orders of magnitude improvement over existing microfluidic approaches.…”

“…While viscoelastic fluids have been shown to have a tunable effect in DLD 5 , we showed both positive and negative contributions to the separation of long (>20 kbp) DNA molecules at high analyte concentrations. An observation we made during these separations at high DNA concentrations or high flow velocities was the emergence of fluctuations in concentration and in the local flow direction that led to decreases in the quality of the separation at concentrations of tens to hundreds of µg/mL 2 . Another example where high DNA concentrations are relevant is for sample preparation for nanopore sequencing (e.g.…”

“…An observation we made during these separations at high DNA concentrations or high flow velocities was the emergence of fluctuations in concentration and in the local flow direction that led to decreases in the quality of the separation at concentrations of tens to hundreds of μg mL −1 . 2 Another example where high DNA concentrations are relevant is for sample preparation for nanopore sequencing ( e.g. Oxford Nanopore: Ligation Sequencing Kit (XL SQK-LSK109-XL) and Rapid Sequencing Kit (SQK-RAD004)).…”

Using symmetry to control viscoelastic waves in pillar arrays

“…Viscoelastic effects in the ows of polymer solutions through and around structures at the microscale are believed to have practical importance in diverse areas ranging from the extraction of oil from mineral matrices 1 and the transport of heat, 2 to the understanding of the transport of biological uids, both in their native environments 3 and through novel types of microuidic devices. 4,5 An example of the latter is microuidics devices for the separation of DNA molecules by length that use deterministic lateral displacement (DLD) [6][7][8][9][10][11] as well those relying on pulsed electrical elds combined with a pillar array arranged in a hexagonal pattern. 12,13 While the underlying mechanisms are entirely different, both are based on arrays of pillars through which the molecules follow size-dependent trajectories.…”