Isolation of target DNA using synergistic magnetic bead transport and electrokinetic flow

Abstract

The advent and dissemination of next-generation sequencing (NGS) technologies such as Illumina's sequencing platforms has brought forth vast reductions in the cost, time, and technical difficulties associated with DNA and RNA sequencing. Despite this trend, the workflow required to generate nucleic acid libraries for sequencing remains time-consuming and laborious. The following research proposes a method for simplifying and streamlining this process by replacing the manual washing steps of the common magnetic bead-based cleanup with a novel microfluidic method by integrating magnetic separation and electrokinetic purification (MSEP). Requiring no pumps, pipette mixing, vortexing, or centrifugation, MSEP relies on selective adsorption of target DNA onto the magnetic beads with subsequent transport of beads through a microchannel undergoing an antiparallel electroosmotic flow. The synergetic flow conditions were optimized using a simple electrohydrodynamic flow model. This work demonstrates that MSEP is as effective in eliminating adapter-dimers from the post-ligation library mix as the manual method while also greatly reducing the hands-on time and amount of pipetting required. Although MSEP has been applied specifically toward NGS library preparation at this time, it has the potential to be adapted and employed for any bead-based separation scheme, namely, solid phase extraction, sequence-specific hybridization, and immunoprecipitation on a microscale.

Automated centrifugal microfluidic system for the preparation of adaptor-ligated sequencing libraries

“…26 Alternatively, magnetic force and electroosmotic flow in opposing directions have been used to separate DNA-bound paramagnetic beads from the supernatant. 27 While these approaches can circumvent the limitations of some microfluidic platforms in handling complex liquid transfer steps, such modifications may require extensive optimization, must be fully compatible with other functions including PCR amplification, and ought to demonstrate robustness with pre-programmed settings.…”

On-Chip Nucleic Acid Purification Followed by ddPCR for SARS-CoV-2 Detection

“…Their work emphasized the following fact: nucleic acid purification is a prerequisite to achieve reliable and accurate detection [5], removal of inhibitors is still required [6], and it increases detection reliability by avoiding false results [7]. However, the present purification process is manual and time-consuming [8], requiring extensive washes with pipette mixing [9], and therefore needs to be implemented carefully to avoid contamination [10,11].…”

Leveraging the fundamentals of heat transfer and fluid mechanics in microscale geometries for automated next-generation sequencing library preparation

“…NGS is a markedly valuable tool for diagnostics and discovery because it generates millions of reads and is exceedingly sensitive to nucleic acid mutations [9][10][11][12] . Unfortunately, while innovations in sequencing technology have reduced requisite cost, time, and workload, sample (library) preparation has largely remained rudimentary and labor-intensive [13][14][15][16] . Even for applications requiring less data, such as microbial whole genome sequencing (WGS) or RNA-seq, the majority of the total sequencing cost can be attributed to library preparation,…”

“…Consequently, expensive robotic liquid handlers dominate research and clinical applications in core laboratories, and small laboratories suffer from the bottleneck of labor-intensive manual protocols. Automation innovation for small laboratories is necessary to accelerate NGS-based research, eliminate sources of human error, and reduce hands-on time [13][14][15][16] . Our previous work started with the innovative concept of integrating liquid mixing steps with thermodynamically driven steps such as ligation and amplification via a single capillary tube heating system [13][14][15][16] .…”

“…Automation innovation for small laboratories is necessary to accelerate NGS-based research, eliminate sources of human error, and reduce hands-on time [13][14][15][16] . Our previous work started with the innovative concept of integrating liquid mixing steps with thermodynamically driven steps such as ligation and amplification via a single capillary tube heating system [13][14][15][16] . This work utilized magnetic bead motion through a microchannel for purification step, as needed by steps C and D in Fig.…”