DNA Sizing with pg/μL Sensitivity for Cell-Free DNA Analysis with the μLAS Technology.

The μLAS technology enables in-line DNA concentration and separation in a microchannel. Here, we describe its operation to analyze the size profile of cell-free DNA (cfDNA) extracted from blood plasma. Operated on commercial systems for capillary electrophoresis, we provide the size distribution of healthy individuals or patients using an input of 10 μL.

Circadian rhythm and circulating cell-free DNA release on healthy subjects.

In the last decade, clinical studies have investigated the clinical relevance of circulating cell-free-DNA (ccfDNA) as a diagnostic and prognosis tool in various diseases including cancers. However, limited knowledge on ccfDNA biology restrains its full development in the clinical practice. To improve our understanding, we evaluated the impact of the circadian rhythm on ccfDNA release in healthy subjects over a 24-h period.

Size and Concentration of Cell-Free DNA Measured Directly from Blood Plasma, without Prior DNA Extraction.

Cell-free DNA in human blood plasma (cfDNA) is now widely used and studied as a biomarker for several physiological and pathological situations. In addition to genetic and epigenetic alterations that provide information about the presence and the nature of non-constitutive DNA in the body, cfDNA concentration and size distribution may potentially be independent biomarkers suitable for monitoring at-risk patients and therapy efficacy. Here, we describe a simple, in-line, method, which measures cfDNA concentration and size distribution from only a few microliters of plasma without the need to extract and/or concentrate the DNA prior to the analysis.

Characterization of Plasma Cell-Free DNA Integrity Using Droplet-Based Digital PCR: Toward the Development of Circulating Tumor DNA-Dedicated Assays.

Cellular-cell free-DNA (ccfDNA) is being explored as a diagnostic and prognostic tool for various diseases including cancer. Beyond the evaluation of the ccfDNA mutational status, its fragmentation has been investigated as a potential cancer biomarker in several studies. However, probably due to a lack of standardized procedures dedicated to preanalytical and analytical processing of plasma samples, contradictory results have been published.

A tunable filter for high molecular weight DNA selection and linked-read sequencing.

In third generation sequencing, the production of quality data requires the selection of molecules longer than ∼20 kbp, but the size selection threshold of most purification technologies is smaller than this target. Here, we describe a technology operated in a capillary with a tunable selection threshold in the range of 3 to 40 kbp controlled by an electric field. We demonstrate that the selection cut-off is sharp, the purification yield is high, and the purification throughput is scalable.

μLAS technology for DNA isolation coupled to Cas9-assisted targeting for sequencing and assembly of a 30 kb region in plant genome.

Cas9-assisted targeting of DNA fragments in complex genomes is viewed as an essential strategy to obtain high-quality and continuous sequence data. However, the purity of target loci selected by pulsed-field gel electrophoresis (PFGE) has so far been insufficient to assemble the sequence in one contig. Here, we describe the μLAS technology to capture and purify high molecular weight DNA.

BIABooster: Online DNA Concentration and Size Profiling with a Limit of Detection of 10 fg/μL and Application to High-Sensitivity Characterization of Circulating Cell-Free DNA.

We describe a technology to perform sizing and concentration analysis of double stranded DNA with a sensitivity of 10 fg/μL in an operating time of 20 min. The technology is operated automatically on a commercial capillary electrophoresis instrument using electro-hydrodynamic actuation. It relies on a new capillary device that achieves online concentration of DNA at the junction between two capillaries of different diameters, thanks to viscoelastic lift forces.

G-quadruplex aptamer selection using capillary electrophoresis-LED-induced fluorescence and Illumina sequencing.

One of the major difficulties that arises when selecting aptamers containing a G-quadruplex is the correct amplification of the ssDNA sequence. Can aptamers containing a G-quadruplex be selected from a degenerate library using non-equilibrium capillary electrophoresis (CE) of equilibrium mixtures (NECEEM) along with high-throughput Illumina sequencing? In this article, we present some mismatches of the G-quadruplex T29 aptamer specific to thrombin, which was PCR amplified and sequenced by Illumina sequencing. Then, we show the proportionality between the number of sequenced molecules of T29 added to the library and the number of sequences obtained in Illumina sequencing, and we find that T29 sequences from this aptamer can be detected in a random library of ssDNA after the sample is fractionated by NECEEM, amplified by PCR, and sequenced.

ssDNA degradation along capillary electrophoresis process using a Tris buffer.

Tris-Acetate buffer is currently used in the selection and the characterization of ssDNA by capillary electrophoresis (CE). By applying high voltage, the migration of ionic species into the capillary generates a current that induces water electrolysis. This phenomenon is followed by the modification of the pH and the production of Tris derivatives.

Aminodextran containing magnetite nanoparticles for molecular biology applications: preparation and evaluation.

Within the framework of medical diagnosis, the main objective is the development of hydrophilic magnetic particles for the generic capture of the nucleic acids in order to enhance the sensitivity. The strategy used in this work is based on the synthesis of cationic and hydrophilic magnetic nanoparticles bearing aminodextran. The synthesis was performed using two different processes: (i) coprecipitation of the ferrous and ferric salts in the presence of an aqueous solution of aminodextran and (ii) via adsorption of aminodextran on iron oxide nanoparticles.

Opto-electronic DNA chip-based integrated card for clinical diagnostics.

Clinical diagnostics is one of the most promising applications for microfluidic lab-on-a-chip or lab-on-card systems. DNA chips, which provide multiparametric data, are privileged tools for genomic analysis. However, automation of molecular biology protocol and use of these DNA chips in fully integrated systems remains a great challenge.

Electrical detection of DNA hybridization based on enzymatic accumulation confined in nanodroplets.

Electrical monitoring of DNA hybridization is one way to reduce the cost and size of the DNA chip reader in comparison with the more classical optical detection. Within electrical methods, electrochemical detection shows very high performances in terms of accuracy and sensitivity, especially when an enzymatic accumulation is used to amplify the signal. However, signal multiplexing for miniaturized systems based on both enzymatic accumulation and electrochemical detection remains challenging due to the Brownian diffusion of the detected product of the enzymatic reaction.

Opto-electronic DNA chip: high performance chip reading with an all-electric interface.

Reading of DNA chips is usually based on fluorescence labeling of hybridised target molecules. Combined with the use of confocal fluorescence scanners, this approach shows very high performances in terms of accuracy and sensitivity. However, fluorescence readers remain costly and cumbersome.

A new generation of scanners for DNA chips.

Today, most of the DNA chips are used with fluorescent markers. Associated with fluorescence confocal scanners, this technology achieves remarkable performances in terms of sensitivity and accuracy. The main technical issues related to these scanners have already been reviewed.