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. By injecting ten times by capillary electrophoresis ssDNA (50 nM), the whole oligonucleotide was degraded. In this paper, we will show that the Tris buffer in the running vials is modified along the electrophoretic process by electrochemical reactions. We also observed that the composition of the metal ions changes in the running buffer vials. This phenomenon, never described in CE, is important for fluorescent ssDNA analysis using Tris buffer. The oligonucleotides are degraded by electrochemically synthesized species (present in the running Tris vials) until it disappears, even if the separation buffer in the capillary is clean. To address these issues, we propose to use a sodium phosphate buffer that we demonstrate to be electrochemically inactive

Clinical relevance of cell-free DNA quantification and qualification during the first month after lung transplantation

BackgroundMany studies have reported the relevance of donor-derived cfDNA (dd-cfDNA) after lung transplantation (LTx) to diagnose and monitor acute rejection (AR) or chronic rejection or infection (INF). However, the analysis of cfDNA fragment size has not been studied. The aim of this study was to determine the clinical relevance of dd-cfDNA and cfDNA size profiles in events (AR and INF) during the first month after LTx.MethodsThis prospective, single-center study includes 62 LTx recipients at the Marseille Nord Hospital, France. Total cfDNA quantification was performed by fluorimetry and digital PCR, dd-cfDNA by NGS (AlloSeq cfDNA-CareDX®), and the size profile by BIABooster (Adelis®). A bronchoalveolar lavage and transbronchial biopsies at D30 established the following groups: not-injured and injured graft (AR, INF, or AR+INF).ResultsQuantification of total cfDNA was not correlated with the patient’s status at D30. The percentage of dd-cfDNA was significantly higher for injured graft patients at D30 (p=0.0004). A threshold of 1.72% of dd-cfDNA correctly classified the not-injured graft patients (negative predictive value of 91.4%). Among recipients with dd-cfDNA >1.72%, the quantification of small sizes (80-120bp) >3.70% identified the INF with high performance (specificity and positive predictive value of 100%).ConclusionWith the aim of considering cfDNA as a polyvalent non-invasive biomarker in transplantation, an algorithm combining the quantification of dd-cfDNA and small sizes of DNA may significantly classify the different types of allograft injuries

Pulsed lasers versus continuous light sources in capillary electrophoresis and fluorescence detection studies: Photodegradation pathways and models

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A comparative study of LED-induced fluorescence and laser-induced fluorescence in SDS-CGE: Application to the analysis of antibodies

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

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DNA separation and enrichment using electro-hydrodynamic bidirectional flows in viscoelastic liquids

International audienceDNA size separation followed by purification and enrichment constitute essential operations for genetic engineering. These processes are mostly carried out using DNA electrophoresis in gels or in polymer solutions , a well-established yet lengthy technique which has been notably improved using Lab-on-Chip technologies. So far, innovations for DNA separation or enrichment have been mostly undertaken separately, and we present an approach that allows us to perform these two processes simultaneously for DNA fragments spanning 0.2–50 kilo base pairs (kbp) in length. Our technology involves an electric field and a counter hydrodynamic flow in viscoelastic liquids, in which we show the occurrence of transverse forces oriented toward the walls. These forces increase with DNA molecular weight (MW) and hence induce a progressive reduction in DNA migration speed that triggers size separation in microfluidic channels as well as in capillaries. The separation of MW markers in the range 1–50 kbp is achieved in 15 minutes, thus outperforming gel electrophoresis that takes ∼3 hours for this sample. Furthermore, the use of a funnel, where electric and flow fields are modulated spatially, enables us to adjust the transverse forces so as to stall the motion of DNA molecules at a position where they accumulate at factors of up to 1000 per minute. In this configuration, we establish that the operations of DNA enrichment and separation can be carried out simultaneously for the bands of a DNA MW marker between 0.2–1.5 kbp diluted at 0.02 ng μL −1 in 30 s. Altogether, our technology, which can readily be integrated as an in-line module in Lab-on-Chips, offers unique opportunities for sample preparation and analysis of minute genomic samples

Determination of free amino acids in African gourd seed milks by capillary electrophoresis with light-emitting diode induced fluorescence and laser-induced fluorescence detection

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Long circulating-free DNA fragments predict early-progression (EP) and progression-free-survival (PFS) in advanced carcinoma treated with immune-checkpoint inhibition (ICI): a new biomarker

International audienceBackgroundEstablishing reliable and early predictive biomarkers of response of ICI is essential. Analysis of cfDNA fragmentation profiles (fragmentome) is a promising non-invasive method to do so independently of a specific molecular target, cancer type or treatment. We monitored plasmatic cfDNA concentration and size characteristics of the fragmentome in advanced lung, head and neck, kidney and bladder cancer patients, treated with ICI (n = 111). The aim was to predict EP (defined as progression at the first imaging evaluation) and PFS.MethodsOur novel patented technology made possible to measure accurately cfDNA concentration and size profile directly from tens of microlitres of plasma and without prior DNA extraction (BIABooster system). Statistical association and predictive performances of response from fragmentome-derived metrics (e.g., concentration, size distribution peaks or fragments size ranges) were conducted. The data was split between a training (n=78) and a test (n=33) set. Optimal thresholds were determined through receiver-operator characteristics (ROC) curve analysis, and confidence intervals determined using bootstrap resampling. Classification metrics were assessed in both the training and testing set. The entire process was bootstrapped 100 times to assess the robustness.ResultsQuantity of long fragments over 1650 bp (LF) showed the best discriminatory power (AUC = 0.77 (0.65-0.87)) of EP. LF were significantly, strongly and positively associated with non-EP (odd ratio =0.27 (0.14-0.52), p <0.001) and longer PFS (p<0.001, hazard ration 0.406 (0.274 - 0.599)). The predictive performances of EP were also very high: AUC 0.75 (0.65-0.84), accuracy 71% (95% CI: 63% - 80%), positive predictive value was 0.61 (0.47-0.78), on the test set.ConclusionsThese findings highlight a very significant association of cfDNA high-molecular-weight fragments with EP and PFS that outperform the predictive value of the only routinely used marker PDL1

Circulating cell-free DNA size distribution as a prediction marker for early progression undergoing immune checkpoint inhibitors

International audienceIntroduction: Despite a significant proportion (20 to 40%)[1] of advanced cancer patients having long-term response to treatment with immune checkpoint inhibitors (ICI), many of them do not respond at all and experience early progression (EP), defined as progression at the first imaging evaluation. Establishing reliable and early predictive biological markers for guiding clinical practice is essential. Analysis of circulating cell-free DNA (cfDNA) fragments size distribution profiles (fragmentome)[2] offer a promising non-invasive method for assessing treatment response independently of a specific molecular target, cancer type, and treatment.Objectives: The SChISM (Size CfDNA Immunotherapies Signature Monitoring) clinical study focuses on monitoring plasmatic cfDNA size profiles, aiming to early adjust therapy to prevent ICI-related progression or toxicity.Methods: We performed statistical and survival biomarker analysis to predict EP and progression-free survival (PFS) using pre-treatment data. Using BIABooster analysis technology from ADELIS, plasmatic fragmentome-derived metrics, including concentration, size location of first and second peaks, and specific size ranges (p = 11 variables), were evaluated alongside standard clinical markers such as performance status, age, and pathology. Predictive analysis of EP was performed by splitting between a training (n = 97) and test (n = 42) set, using stratified sampling, Optimal thresholds were determined on the training dataset through receiver-operator characteristics (ROC) curve analysis, and confidence intervals determined using bootstrap resampling. Classification metrics were assessed in both the training and testing set. The entire process was bootstrapped 100 times to assess the robustness of the results. Empirical longitudinal models were developed to capture both inter- and intra-patient variability in cfDNA metrics.Results: Analyses were performed on a cohort of 139 advancer or metastatic cancer patients (melanoma, clear cell kidney cancer, urothelial bladder carcinoma, squamous cell carcinoma of the head and neck or non-small cell lung cancer) treated with monoclonal antibodies in monotherapy or in combination. Three out of the eleven features were associated with EP and/or PFS. The relative quantity of fragments over 1650 base pairs (bp) (OVER_1650) exhibited the highest discriminatory power for EP (median area under ROC curve (AUC) on the test set = 0.7, 95% CI: 0.59-0.80). A lower relative quantity of these long fragments correlated with EP in both the univariate (odd ratio OR = 0.37 (0.22-0.64), p < 0.001) and multivariate (OR = 0.45 (0.25-0.81), p = 0.008) settings. Additionally, all cfDNA metrics except the first peak’s location were better estimated by empirical non-constant models, highlighting significant variability in cfDNA quantities over time.Conclusions: These findings highlight the association of high-molecular-weight fragments with the early progressors ICI-treated. Thus, the improvement of the knowledge in the long fragments’ biology, as well as the understanding of the cfDNA release over time, could potentially help in preventing or discontinuing unnecessary treatments for patients who are unlikely to respond to ICI

Long circulating-free DNA fragments predict early-progression (EP) and progression-free-survival (PFS) in advanced carcinoma treated with immune-checkpoint inhibition (ICI): a new biomarker

International audienceBackgroundEstablishing reliable and early predictive biomarkers of response of ICI is essential. Analysis of cfDNA fragmentation profiles (fragmentome) is a promising non-invasive method to do so independently of a specific molecular target, cancer type or treatment. We monitored plasmatic cfDNA concentration and size characteristics of the fragmentome in advanced lung, head and neck, kidney and bladder cancer patients, treated with ICI (n = 111). The aim was to predict EP (defined as progression at the first imaging evaluation) and PFS.MethodsOur novel patented technology made possible to measure accurately cfDNA concentration and size profile directly from tens of microlitres of plasma and without prior DNA extraction (BIABooster system). Statistical association and predictive performances of response from fragmentome-derived metrics (e.g., concentration, size distribution peaks or fragments size ranges) were conducted. The data was split between a training (n=78) and a test (n=33) set. Optimal thresholds were determined through receiver-operator characteristics (ROC) curve analysis, and confidence intervals determined using bootstrap resampling. Classification metrics were assessed in both the training and testing set. The entire process was bootstrapped 100 times to assess the robustness.ResultsQuantity of long fragments over 1650 bp (LF) showed the best discriminatory power (AUC = 0.77 (0.65-0.87)) of EP. LF were significantly, strongly and positively associated with non-EP (odd ratio =0.27 (0.14-0.52), p <0.001) and longer PFS (p<0.001, hazard ration 0.406 (0.274 - 0.599)). The predictive performances of EP were also very high: AUC 0.75 (0.65-0.84), accuracy 71% (95% CI: 63% - 80%), positive predictive value was 0.61 (0.47-0.78), on the test set.ConclusionsThese findings highlight a very significant association of cfDNA high-molecular-weight fragments with EP and PFS that outperform the predictive value of the only routinely used marker PDL1