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

Author

Geoffroy Poulet, Jean-Sébastien Hulot, Anne Blanchard, Damien Bergerot, Wenjin Xiao, Frederic Ginot, Audrey Boutonnet-Rodat, Abdelli Justine, Guillaume Beinse, Vanna Geromel, Laurence Pellegrina, Michel Azizi, Pierre Laurent-Puig, Leonor Benhaim, and Valerie Taly

Subjects
Medicine, Science
Abstract

Abstract 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. 10 healthy female subjects underwent blood sampling at 8am and 20 healthy male subjects underwent serial blood sampling (8:00 AM, 9:00 AM, 12:00 PM, 4:00 PM, 8:00 PM, 12:00 AM, 4 AM (+ 1 Day) and 8 AM (+ 1 Day)). We performed digital droplet-based PCR (ddPCR) assays to target 2 DNA fragments (69 & 243 bp) located in the KRAS gene to determine the ccfDNA concentration and fragmentation profile. As control, half of the samples were re-analyzed by capillary miniaturized electrophoresis (BIAbooster system). Overall, we did not detect any influence of the circadian rhythm on ccfDNA release. Instead, we observed a decrease in the ccfDNA concentration after meal ingestion, suggesting either a post-prandial effect or a technical detection bias due to a higher plasma load in lipids and triglycerides. We also noticed a potential effect of gender, weight and creatinine levels on ccfDNA concentration.

Published
2023
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2. Characterization of Plasma Cell-Free DNA Integrity Using Droplet-Based Digital PCR: Toward the Development of Circulating Tumor DNA-Dedicated Assays

Author

Geoffroy Poulet, Fanny Garlan, Sonia Garrigou, Eleonora Zonta, Leonor Benhaim, Marie-Jennifer Carrillon, Audrey Didelot, Delphine Le Corre, Claire Mulot, Philippe Nizard, Frederic Ginot, Audrey Boutonnet-Rodat, Helene Blons, Jean-Baptiste Bachet, Julien Taïeb, Aziz Zaanan, Vanna Geromel, Laurence Pellegrina, Pierre Laurent-Puig, Shu-Fang Wang-Renault, and Valerie Taly

Subjects
circulating tumor DNA, picoliter-droplet digital PCR, cancer biomarker, apoptosis, necrosis, DNA integrity index, Neoplasms. Tumors. Oncology. Including cancer and carcinogens, RC254-282
Abstract

Background: 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.Methods: ddPCR assays allowing the detection of KRAS wild-type and mutated sequences (KRAS p.G12V, pG12D, and pG13D) were designed to target different fragments sizes. Once validated on fragmented and non-fragmented DNA extracted from cancer cell lines, these assays were used to investigate the influence of the extraction methods on the non-mutated and mutated ccfDNA integrity reflected by the DNA integrity index (DII). The DII was then analyzed in two prospective cohorts of metastatic colorectal cancer patients (RASANC study n = 34; PLACOL study n = 12) and healthy subjects (n = 49).Results and Discussion: Our results demonstrate that ccfDNA is highly fragmented in mCRC patients compared with healthy individuals. These results strongly suggest that the characterization of ccfDNA integrity hold great promise toward the development of a universal biomarker for the follow-up of mCRC patients. Furthermore, they support the importance of standardization of sample handling and processing in such analysis.

Published
2021
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3. A tunable filter for high molecular weight DNA selection and linked-read sequencing

Author

Adrien Castinel, Frederic Ginot, Aurélien Bancaud, Audrey Gibert, Olivier Bouchez, Géraud Bouwmans, Juan-Luis Fuentes Rojas, Cécile Donnadieu, Audrey Boutonnet, Laurent Bigot, Nicolas Milon, Karen Baudelle, Équipe Micro-Nanofluidique pour les sciences de la vie et de l’environnement (LAAS-MILE), Laboratoire d'analyse et d'architecture des systèmes (LAAS), Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées, Génome et Transcriptome - Plateforme Génomique ( GeT-PlaGe), Plateforme Génome & Transcriptome (GET), Génopole Toulouse Midi-Pyrénées [Auzeville] (GENOTOUL), Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Génopole Toulouse Midi-Pyrénées [Auzeville] (GENOTOUL), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Laboratoire de Physique des Lasers, Atomes et Molécules - UMR 8523 (PhLAM), Université de Lille-Centre National de la Recherche Scientifique (CNRS), Picometrics Technologies, ANR-16-CE18-0028,MicroLAS,µ-Laboratoire d'Analyse et de Séparation des chromosomes : développement d'un outil pour le typage rapide des bactéries, levures et cellules de mammifères(2016), ANR-16-ASTR-0020,BIOPULSE,Dispositif de détection biologique ultrasensible de pathogènes prélevés par air pulsé.(2016), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J), Institut National de la Recherche Agronomique (INRA), Laboratoire de Génétique Cellulaire (LGC), Ecole Nationale Vétérinaire de Toulouse (ENVT), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA), Photonique (Photonique), Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université Toulouse - Jean Jaurès (UT2J), Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT), Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Ecole Nationale Vétérinaire de Toulouse (ENVT), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Génopole Toulouse Midi-Pyrénées [Auzeville] (GENOTOUL), and Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)

Subjects
Computer science, Sequencing data, Biomedical Engineering, Bioengineering, 02 engineering and technology, Third generation sequencing, Computational biology, Biochemistry, [SPI.MAT]Engineering Sciences [physics]/Materials, 03 medical and health sciences, Cucumis melo, Particle Size, [PHYS.COND]Physics [physics]/Condensed Matter [cond-mat], Throughput (business), ComputingMilieux_MISCELLANEOUS, Selection (genetic algorithm), 030304 developmental biology, 0303 health sciences, DNA, Sequence Analysis, DNA, General Chemistry, 021001 nanoscience & nanotechnology, Molecular Weight, genomic DNA, Filter (video), [SPI.OPTI]Engineering Sciences [physics]/Optics / Photonic, 0210 nano-technology, Block size, High molecular weight dna
Abstract

International audience; 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. We also provide an analytical model that the actuation settings of the filter. The selection of high molecular weight genomic DNA from the melon Cucumis melo L., a diploid organism of ∼0.45 Gbp, is then reported. Linked-read sequencing data show that the N50 phase block size, which scores the correct representation of two chromosomes, is enhanced by a factor of 2 after size selection, establishing the relevance and versatility of our technology.

Published
2020
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4. Circadian Rhythm and Circulating Cell-Free DNA Release on Healthy Subjects

Author

Geoffroy Poulet, Jean-Sébastien Hulot, Anne Blanchard, Damien Bergerot, Wenjin Xiao, Frederic Ginot, Audrey Boutonnet-Rodat, Guillaume Beinse, Vanna Geromel, Laurence Pellegrina, Michel Azizi, Pierre Laurent-Puig, Leonor Benhaim, and Valerie Taly

Abstract

Background 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-hour period. Methods 20 healthy male subjects underwent serial blood sampling (8:00 AM, 9:00 AM, 12:00 AM, 4:00 PM, 8:00 PM, 12:00 PM, 4 AM (+1 Day) and 8 AM (+1 Day)). We performed digital droplet-based PCR (ddPCR) assays to target 2 DNA fragments (69 & 243 bp) located in the KRAS gene to determine the ccfDNA concentration and fragmentation profile. For control, half of the samples were re-analyzed by capillary miniaturized electrophoresis (BIAbooster system). Results & discussion Overall, we did not detect any influence of the circadian rhythm on ccfDNA release. Instead, we observed a decrease in the ccfDNA concentration after meal ingestion, suggesting either a post-prandial effect or a technical detection bias due to a higher plasma load in lipids and triglycerides. We also noticed a potential effect of gender, weight and creatinine levels on the ccfDNA concentration.

Published
2022
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5. 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

Author

Valérie Taly, Aurélien Bancaud, Audrey Didelot, Fanny Garlan, Sonia Garrigou, Comtet-Louis Andriamanampisoa, Frederic Ginot, Audrey Boutonnet-Rodat, Daniel Henaff, Jacques Fabre, Laure Saias, Arnaud Morin, Frédéric Fina, Vincent Picot, Aziz Zaanan, Pierre Laurent-Puig, Caroline Gaudy, Pascale Tomasini, Picometrics Technologies, Équipe Micro-Nanofluidique pour les sciences de la vie et de l’environnement (LAAS-MILE), Laboratoire d'analyse et d'architecture des systèmes [Toulouse] (LAAS), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UPS), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UPS), Université Fédérale Toulouse Midi-Pyrénées, Médecine Personnalisée, Pharmacogénomique, Optimisation Thérapeutique (MEPPOT - U1147), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris Descartes - Paris 5 (UPD5), Centre de Recherches en Oncologie biologique et Oncopharmacologie (CRO2), Aix Marseille Université (AMU)- Hôpital de la Timone [CHU - APHM] (TIMONE)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire des Sciences de l'Information et des Systèmes (LSIS), Aix Marseille Université (AMU)-Université de Toulon (UTLN)-Arts et Métiers Paristech ENSAM Aix-en-Provence-Centre National de la Recherche Scientifique (CNRS), HEMODIA, Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), Laboratoire d'Innovation pour les Technologies des Energies Nouvelles et les nanomatériaux (LITEN), Institut National de L'Energie Solaire (INES), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Savoie Mont Blanc (USMB [Université de Savoie] [Université de Chambéry])-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Savoie Mont Blanc (USMB [Université de Savoie] [Université de Chambéry])-Centre National de la Recherche Scientifique (CNRS), Physico-Chimie-Curie (PCC), Centre National de la Recherche Scientifique (CNRS)-Institut Curie-Université Pierre et Marie Curie - Paris 6 (UPMC), Service d'Oncologie Multidisciplinaire et d'Innovations Thérapeutiques, Nord University Hospital of Marseille, Laboratoire d'analyse et d'architecture des systèmes (LAAS), Université Toulouse Capitole (UT Capitole), Institut National des Sciences Appliquées (INSA)-Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées (INSA)-Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse Capitole (UT Capitole), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL), Université de Picardie Jules Verne (UPJV), Laboratoire de Transfert d'Oncologie Biologique [Hôpital Nord - APHM], Aix Marseille Université (AMU)-Assistance Publique - Hôpitaux de Marseille (APHM)- Hôpital Nord [CHU - APHM], Institut de Génétique Moléculaire de Montpellier (IGMM), Unicancer, Institut Curie [Paris], Laboratoire des Composants PEM (LCPEM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Curie [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche des Cordeliers (CRC (UMR_S_1138 / U1138)), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), TALY, Valerie, Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université Toulouse - Jean Jaurès (UT2J), Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT), École Pratique des Hautes Études (EPHE), Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Hôpital Nord [CHU - APHM]-Assistance Publique - Hôpitaux de Marseille (APHM)-Aix Marseille Université (AMU), Centre National de la Recherche Scientifique (CNRS)-Arts et Métiers Paristech ENSAM Aix-en-Provence-Université de Toulon (UTLN)-Aix Marseille Université (AMU), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), and Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC)

Subjects
0301 basic medicine, Accuracy and precision, Capillary action, [SDV]Life Sciences [q-bio], [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Polymerase Chain Reaction, Analytical Chemistry, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Capillary electrophoresis, Limit of Detection, Neoplasms, Biomarkers, Tumor, Humans, Digital polymerase chain reaction, Detection limit, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, Chromatography, Dna concentration, Electrophoresis, Capillary, Equipment Design, Circulating Cell-Free DNA, [SDV] Life Sciences [q-bio], 030104 developmental biology, chemistry, 030220 oncology & carcinogenesis, Hydrodynamics, Cell-Free Nucleic Acids, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, DNA
Abstract

International audience; 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. Using a set of DNA ladders in the range of 100–1500 bp, we report a sizing accuracy and precision better than 3% and a concentration quantification precision of ∼20%. When the technology is applied to the analysis of clinical samples of circulating cell-free DNA (cfDNA), the measured cfDNA concentrations are in good correlation with those measured by digital PCR. Furthermore, the cfDNA size profiles indicate that the fraction of low molecular weight cfDNA in the range of 75–240 bp is a candidate biomarker to discriminate between healthy subjects and cancer patients. We conclude that our technology is efficient in analyzing highly diluted DNA samples and suggest that it will be helpful in translational and clinical research involving cfDNA.

Published
2018
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6. μLAS technology for DNA isolation coupled to Cas9-assisted targeting for sequencing and assembly of a 30 kb region in plant genome

Author

Aurélien Bancaud, Carine Satge, Tslil Gabrieli, Sandra Moreau, Caroline Callot, Audrey Boutonnet, Arnaud Bellec, Hélène Bergès, Margaux-Alison Fustier, Nicolas Milon, Stéphane Cauet, Laure Saias, Céline Chantry-Darmon, Frederic Ginot, Équipe Micro-Nanofluidique pour les sciences de la vie et de l’environnement (LAAS-MILE), Laboratoire d'analyse et d'architecture des systèmes (LAAS), Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université Toulouse - Jean Jaurès (UT2J), Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT), Adelis Technologies, Centre National de Ressources Génomiques Végétales (CNRGV), Institut National de la Recherche Agronomique (INRA), Laboratoire des interactions plantes micro-organismes (LIPM), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS), Tel Aviv University (TAU), Picometrics Technologies, European Commission Joint Research Centre, Institut Carnot, Region Auvergne-Rhone-Alpes, Region Bourgogne-Franche-Comte, Region Hauts-de-France, Region Nouvelle-Aquitaine, ANR-16-CE18-0028,MicroLAS,µ-Laboratoire d'Analyse et de Séparation des chromosomes : développement d'un outil pour le typage rapide des bactéries, levures et cellules de mammifères(2016), Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées, Tel Aviv University [Tel Aviv], Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J), Laboratoire d'Analyse Génétique pour les Espèces Animales (LABOGENA), Université Paris-Sud - Paris 11 (UP11), and Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)

Subjects
Chromosomes, Artificial, Bacterial, DNA, Plant, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Locus (genetics), 02 engineering and technology, Computational biology, Biology, Genome, 03 medical and health sciences, Plasmid, Medicago truncatula, Genetics, 030304 developmental biology, 0303 health sciences, Bacterial artificial chromosome, Contig, Computational Biology, Reproducibility of Results, Sequence Analysis, DNA, Genomics, 021001 nanoscience & nanotechnology, DNA extraction, Electrophoresis, Gel, Pulsed-Field, DNA profiling, CRISPR-Cas Systems, 0210 nano-technology, Genome, Plant, Reference genome
Abstract

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. First, the technology is optimized to perform high sensitivity DNA profiling with a limit of detection of 20 fg/μl for 50 kb fragments and an analytical time of 50 min. Then, μLAS is operated to isolate a 31.5 kb locus cleaved by Cas9 in the genome of the plant Medicago truncatula. Target purification is validated on a Bacterial Artificial Chromosome plasmid, and subsequently carried out in whole genome with μLAS, PFGE or by combining these techniques. PacBio sequencing shows an enrichment factor of the target sequence of 84 with PFGE alone versus 892 by association of PFGE with μLAS. These performances allow us to sequence and assemble one contig of 29 441 bp with 99% sequence identity to the reference sequence.

Published
2019
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7. Elaboration of hydrophilic aminodextran containing submicron magnetic latex particles

Author

Sebastian Braconnot, Hanna Mouaziz, Abdelhamid Elaissari, and Frederic Ginot

Subjects
Polymers and Plastics, Chemistry, Radical polymerization, Emulsion polymerization, equipment and supplies, Colloid, Colloid and Surface Chemistry, Adsorption, Chemical engineering, Polymerization, Emulsion, Polymer chemistry, Materials Chemistry, Magnetic nanoparticles, Particle size, Physical and Theoretical Chemistry, human activities
Abstract

Aminodextran containing submicron magnetic latex particles were prepared in two steps: (a) transformation of oil-in-water magnetic emulsion into structured magnetic latex particles via combination of seed and miniemulsion-like polymerization process and (b) immobilization (adsorption and chemical grafting) of prepared aminodextran onto negatively charged seed magnetic latex particles. The elaborated magnetic latex particles were characterized in terms of particle size, size distribution, morphology, surface charge density, chemical composition, magnetic properties, and also colloidal stability. The results showed that the morphology of the prepared seed magnetic latex is core–shell like and the cationic latex particles are hydrophilic and of high colloidal stability, irrespective of the aminodextran immobilization process.

Published
2009
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8. Opto-electronic DNA chip-based integrated card for clinical diagnostics

Author

Frédéric Mallard, Veronique Lanet, Denis Roller, Frederic Ginot, Bernard Calvas, Gilles Marchand, Raymond Campagnolo, Frédéric Foucault, Patrick Broyer, Cyril Delattre, and Lionel Menou

Subjects
Engineering, business.industry, Reading (computer), Microfluidics, Biomedical Engineering, computer.software_genre, Chip, Automation, Embedded system, Electronic engineering, Computer-Aided Design, Humans, Computer Aided Design, Fluidics, DNA microarray, business, Molecular Biology, computer, Protocol (object-oriented programming), Oligonucleotide Array Sequence Analysis
Abstract

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. Simplicity of chip and/or card/instrument interfaces is amongst the most critical issues to be addressed. Indeed, current detection systems for DNA chip reading are often complex, expensive, bulky and even limited in terms of sensitivity or accuracy. Furthermore, for liquid handling in the lab-on-cards, many devices use complex and bulky systems, either to directly manipulate fluids, or to ensure pneumatic or mechanical control of integrated valves. All these drawbacks prevent or limit the use of DNA-chip-based integrated systems, for point-of-care testing or as a routine diagnostics tool. We present here a DNA-chip-based protocol integration on a plastic card for clinical diagnostics applications including: (1) an opto-electronic DNA-chip, (2) fluid handling using electrically activated embedded pyrotechnic microvalves with closing/opening functions. We demonstrate both fluidic and electric packaging of the optoelectronic DNA chip without major alteration of its electronical and biological functionalities, and fluid control using novel electrically activable pyrotechnic microvalves. Finally, we suggest a complete design of a card dedicated to automation of a complex biological protocol with a fully electrical fluid handling and DNA chip reading.

Published
2007
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9. Opto-electronic DNA chip: high performance chip reading with an all-electric interface

Author

Raymond Campagnolo, Frédéric Mallard, Gilles Marchand, and Frederic Ginot

Subjects
Optics and Photonics, Computer science, Interface (computing), Biomedical Engineering, Biophysics, Photodetector, Nanotechnology, Biosensing Techniques, Multiplexing, Electrochemistry, Oligonucleotide Array Sequence Analysis, business.industry, Dynamic range, Microchemistry, Signal Processing, Computer-Assisted, DNA, Equipment Design, General Medicine, Chip, Equipment Failure Analysis, CMOS, Luminescent Measurements, Optoelectronics, Electronics, DNA microarray, business, Biosensor, Biotechnology
Abstract

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. This prevents the use of DNA chips as a decentralised testing tool. Electrical monitoring of hybridisation is one way to reduce the cost and size of the reader. However, the multiplexing of electric detection-based systems in a miniaturised form remains challenging. Here, we present a system based on the use of a low cost CMOS photodetector array as a solid support for a DNA chip, coupled with revelation by enzyme-catalysed chemiluminescence. This system is shown to allow the detection of low pM target concentrations with a 3 logs dynamic range on dense DNA microarrays, with excellent inter-spot reproducibility. Combining electric interface and high analytical performances, this opto-electronic DNA chip is one attractive solution for nucleic acids detection and analysis in disposable, fully automatised, total analysis systems developed for decentralised testing.

Published
2005
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10. A new generation of scanners for DNA chips

Author

Patrick Pouteau, Pierre Puget, O Peyssonneaux, Frederic Ginot, G McGall, Pierre Labeye, Richard Gonzalez, François Perraut, Alexandre Lagrange, and Lionel Menou

Subjects
Optics and Photonics, Scanner, Computer science, ComputingMethodologies_IMAGEPROCESSINGANDCOMPUTERVISION, Biomedical Engineering, Biophysics, Sensitivity and Specificity, Fluorescence, law.invention, Optics, Optical reader, HIV Protease, law, Electrochemistry, Humans, Oligonucleotide Array Sequence Analysis, Microscopy, Confocal, business.industry, Reproducibility of Results, Equipment Design, General Medicine, Chip, CD player, Lens (optics), Optical format, Feasibility Studies, RNA, Viral, business, Focus (optics), Optical disc, Computer hardware, Biotechnology
Abstract

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. However, these scanners are costly, especially when high density chips are used. In this case, a mechanical precision of 1 mm or less is required to achieve the measurement precision required. This cost level prevents the spread of this technology in the diagnostic market. We will present a new concept for scanners with equivalent or superior performances, with a cost cut of 5/10. This concept is inspired from the field of optical disk and reader. Basically, an optical format is added to the chip, before DNA deposition. This format contains tracks which are superimposed to the DNA features. These tracks define the path that an optical head of a CD player must follow in order to scan the surface of the DNA chip. Such a head is a very cheap component, and has a precision of less than 100 nm thanks to real-time focus and tracking. These functions are fulfilled by electromagnetic actuators mounted on the support of the frontal lens. We show here that it is possible to use such a head to build a fluorescence confocal scanner with equivalent or even better performances than conventional scanners. # 2002 Elsevier Science B.V. All rights reserved.

Published
2002
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11. Aminodextran containing magnetite nanoparticles for molecular biology applications: preparation and evaluation

Author

Frederic Ginot, Abdelhamid Elaissari, Raphael Veyret, Alain Theretz, Hanna Mouaziz, Laboratoire d'automatique et de génie des procédés (LAGEP), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Systèmes Macromoléculaires et Physiopathologie Humaine (SMPH), BIOMERIEUX-Centre National de la Recherche Scientifique (CNRS), Recherche Technologique, bioMerieux SA, and BIOMERIEUX

Subjects
Materials science, Coprecipitation, Macromolecular Substances, Surface Properties, Drug Compounding, Inorganic chemistry, Biomedical Engineering, Molecular Conformation, Pharmaceutical Science, Medicine (miscellaneous), Molecular Probe Techniques, Bioengineering, 02 engineering and technology, 010402 general chemistry, 01 natural sciences, Ferrous, chemistry.chemical_compound, Adsorption, Materials Testing, General Materials Science, [SPI.GPROC]Engineering Sciences [physics]/Chemical and Process Engineering, Particle Size, Molecular Biology, ComputingMilieux_MISCELLANEOUS, Drug Carriers, Aqueous solution, ACL, Cationic polymerization, Dextrans, [SDV.SP]Life Sciences [q-bio]/Pharmaceutical sciences, 021001 nanoscience & nanotechnology, Ferrosoferric Oxide, 0104 chemical sciences, Nanostructures, Chemical engineering, chemistry, Nucleic acid, Magnetic nanoparticles, 0210 nano-technology, Crystallization, Iron oxide nanoparticles
Abstract

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. The obtained particles are characterized and evaluated in non-specific nucleic acids extraction and amplification.

Published
2010
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12. Electrical detection of DNA hybridization based on enzymatic accumulation confined in nanodroplets

Author

Patrick Pouteau, Raymond Campagnolo, Frederic Ginot, Gilles Marchand, and Cyril Delattre

Subjects
Detection limit, Base pair, Chemistry, DNA–DNA hybridization, Hybridization probe, Analytical chemistry, Nucleic Acid Hybridization, Electrochemistry, Analytical Chemistry, Enzymes, Nanostructures, Solutions, Nucleic acid thermodynamics, Chemical engineering, Electricity, Electrode, A-DNA, DNA Probes, Base Pairing, Electrodes, Oligonucleotide Array Sequence Analysis
Abstract

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. We present here a DNA chip with electrical detection based on the following sequence: (i) hybridization of nucleic acids and washing in a liquid layer as usual, (ii) formation of independent nanodroplets on each detection site, (iii) enzymatic accumulation in each droplet avoiding cross-contamination between neighboring sites, and (iv) electrochemical detection of the product accumulated during the enzymatic reaction. The simple and fast transition from the liquid layer (hybridization step) to an array of nanodroplets (enzymatic accumulation and detection steps) was performed through the filling of the hybridization chamber with a solution containing the enzymatic substrates, the drawing of this solution, and the simultaneous creation of droplets thanks to retention areas based on circular rims or hydrophilic rings. Using this approach, hybridization is achieved in a liquid layer as usual, followed by the enzymatic accumulation in nanodroplets to avoid the cross-talk between neighboring sites. Moreover, working in droplets enables a fast increase in the concentration of the product generated by the enzymatic reaction and thus an improvement of the detection limit of the system.

Published
2005

13. Some potential uses of photothermal detection for DNA microarray quantification

Author

Catherine Pelle, Francoise Vinet, Frederic Ginot, Stephane Getin, Pierre Puget, Ludovic Poupinet, Patrick Chaton, and Armelle Novelli

Subjects
Absorbance, Optical absorbance, Optics, Computer science, Monitor quality, business.industry, Digital image processing, Photothermal therapy, DNA microarray, Biological system, business
Abstract

Photothermal deflection technique is a very sensitive mean to measure optical absorbance. This study is aimed at evaluating its potentiality in order to detect hybridation and to monitor quality control. The principle of the technique relies on the fact that nucleic acids present the property to absorb light between 220 nm to 280 nm. A first theoretical approach based on solving light and thermal equations demonstrates the ability of photothermal deflection to detect hybridisation. This point is checked through several experimentations with oligonucleotid with 32 mers targets lengths. An important point to stres is the specificity of the signals obtained and the ability of automation of the reading with image processing algorithms. At last we focus our attention on the ability of this technique to in-situ synthesis process. Our experimental study shows the ability of this characterisation with a detection sensitivity of one base.

Published
2003
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14. Adsorption de protéines à une interface eau/air fonctionnalisée

Author

Jean Berthier, Nicolas Blondiaux, Frederic Ginot, Laurent Davoust, Dominique Masse, Département Microtechnologies pour la Biologie et la Santé (DTBS), Laboratoire d'Electronique et des Technologies de l'Information (CEA-LETI), Université Grenoble Alpes [Saint Martin d'Hères] - Commissariat à l'énergie atomique et aux énergies alternatives (CEA) - Université Grenoble Alpes [Saint Martin d'Hères] - Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire des écoulements géophysiques et industriels (LEGI), Université Joseph Fourier - Grenoble 1 (UJF) - Institut National Polytechnique de Grenoble (INPG) - Centre National de la Recherche Scientifique (CNRS), Institut de Mécanique Céleste et de Calcul des Ephémérides (IMCCE), Université Pierre et Marie Curie - Paris 6 (UPMC) - Université de Lille, Sciences et Technologies - Observatoire de Paris - INSU - Centre National de la Recherche Scientifique (CNRS), Informatique, Biologie Intégrative et Systèmes Complexes (IBISC), Université d'Évry-Val-d'Essonne (UEVE) - Centre National de la Recherche Scientifique (CNRS), Les éléments transposables : du génome aux populations (ETGAP), École normale supérieure - Lyon (ENS Lyon) - École normale supérieure - Paris (ENS Paris) - Institut National de la Recherche Agronomique (INRA) - Université Paris-Sud - Paris 11 (UP11) - Université Pierre et Marie Curie - Paris 6 (UPMC) - Université de Rennes 1 (UR1) - Université Claude Bernard Lyon 1 (UCBL) - Université Blaise Pascal - Clermont-Ferrand 2 (UBP) - Université Paul Sabatier - Toulouse 3 (UPS) - Université Paris Diderot - Paris 7 (UP7) - Université d'Évry-Val-d'Essonne (UEVE) - BIOMERIEUX - Institut National de la Santé et de la Recherche Médicale (INSERM) - Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives - Laboratoire d'Electronique et de Technologie de l'Information (CEA-LETI), Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire des Écoulements Géophysiques et Industriels [Grenoble] (LEGI), Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF), Centre National de la Recherche Scientifique (CNRS)-Université de Lille-Observatoire de Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut national des sciences de l'Univers (INSU - CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC), Département d'Architectures, Conception et Logiciels Embarqués-LETI (DACLE-LETI), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), BIOMERIEUX, Université Joseph Fourier - Grenoble 1 (UJF)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut national des sciences de l'Univers (INSU - CNRS)-Observatoire de Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Université Grenoble Alpes (UGA)-Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Direction de Recherche Technologique (CEA) (DRT (CEA)), Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), PSL Research University (PSL)-PSL Research University (PSL)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), École normale supérieure - Lyon (ENS Lyon)-École normale supérieure - Paris (ENS Paris)-Institut National de la Recherche Agronomique (INRA)-Université Paris-Sud - Paris 11 (UP11)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Blaise Pascal - Clermont-Ferrand 2 (UBP)-Université Toulouse III - Paul Sabatier (UT3), and Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Université Paris Diderot - Paris 7 (UPD7)-Université d'Évry-Val-d'Essonne (UEVE)-BIOMERIEUX-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Microscope, Materials science, 010504 meteorology & atmospheric sciences, [PHYS.MECA.MEFL]Physics [physics]/Mechanics [physics]/Mechanics of the fluids [physics.class-ph], Diffusion, Kinetics, 0207 environmental engineering, [SPI.MECA.MEFL] Engineering Sciences [physics]/Mechanics [physics.med-ph]/Fluids mechanics [physics.class-ph], 02 engineering and technology, [PHYS.MECA.MEFL] Physics [physics]/Mechanics [physics]/Mechanics of the fluids [physics.class-ph], 01 natural sciences, Fluorescence, Finite element method, law.invention, [SPI.MECA.MEFL]Engineering Sciences [physics]/Mechanics [physics.med-ph]/Fluids mechanics [physics.class-ph], Condensed Matter::Soft Condensed Matter, Planar, Adsorption, Chemical engineering, law, [PHYS.MECA.MEFL]Physics [physics]/Mechanics [physics]/Fluid mechanics [physics.class-ph], 020701 environmental engineering, 0105 earth and related environmental sciences, Water Science and Technology
Abstract

International audience; An optical set-up coupled to a microscope is developed in order to follow the aging kinetics of a planar air/water interface from induced fluorescence. The technique is characterized with fluorescent-tagged particles and further applied to an air/water interface functionalized to adsorb selectively strepavidin. The second configuration under study turns out to be a 2-D foam entrapped between two glass plates ; a modelling taking into account diffusion and adsorption is solved by finite element method.

Published
2003
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15. Correction of Some Genotyping Errors in Automated Fluorescent Microsatellite Analysis by Enzymatic Removal of One Base Overhangs

Author

Gabor Gyapay, Frederic Ginot, Isabelle Bordelais, and Simon Nguyen

Subjects
Exonuclease, Genetics, Genome, biology, DNA polymerase, Sequence Analysis, DNA, Molecular biology, Fluorescence, law.invention, chemistry.chemical_compound, chemistry, law, biology.protein, Animals, Humans, Microsatellite, Genotyping, Polymerase chain reaction, Polymerase, DNA, Taq polymerase, Research Article, Microsatellite Repeats
Abstract

The conjunction of high resolution genetic maps based on (CA)n microsatellite markers (1) and fluorescent genotyping (2) has led to research programs which require the determination of hundreds of thousands of genotypes. However, the migration profile of a (CA)n microsatellite marker after PCR (polymerase chain reaction) is often complicated because of slippage of Taq polymerase during PCR, and because of the ability of this polymerase to add an extra base at the end of the amplified fragments in a template dependent manner (3). In the traces generated by a fluorescent sequencer, these phenomena generate spurious bands as shown in Figure 1. These extra bands are usually referred to as ‘shadow bands’ (4). This complicated pattern is relatively easily recognized by an experienced eye, but may mislead the software which automatically determines the sample alleles (we use ABI 373A sequencers with 24 cm gels for the electrophoresis, and the ABI software Genescan and Genotyper for peak sizing and allele calling respectively). Because of this, the operator must check visually all the traces and the allele calls made by the software. This is time-consuming, and prone to human error after a few hundred checks. We attempted therefore to eliminate most of the allele calling errors by eliminating the extra bases added by the Taq polymerase. We tried to do this by using Pfu polymerase either during or after PCR, or by using T4 DNA polymerase after PCR. Pfu polymerase is thermostable and possesses a proofreading activity (3). In some cases, however, the extra base added by the Taq polymerase was not completely removed by this enzyme (Fig. 2A and B). This phenomenon is probably due to competition between the extendase activity of the Taq polymerase and the exonuclease activity of the Pfu polymerase as both enzymes are active at 72 C. On the contrary, and as suggested by others (5), a T4 polymerase treatment, similar to that conventionally used to make blunt double-stranded linear DNA fragments, completely removed the bases added by the Taq polymerase (without exception so far; Fig. 2C). The simplification of the electrophoresis pattern is sometimes spectacular (Fig. 3). The use of Pfu polymerase alone during PCR avoids the problem of the extra base addition (3), and was used successfully in fluorescent genotyping (6). However, PCR efficiency is lower than when using Taq polymerase, making it necessary to precipitate the samples during multiplex analysis (data not shown). Figure 1. Typical electrophoresis pattern of a (CA)n microsatellite marker with shadow bands. The two alleles of this heterozygous sample are eight bases apart, and are shown with their estimated size. The rightmost peak of each group corresponds to an extra base addition (peaks labelled +1). On the left, we see peaks corresponding to slippage of the Taq polymerase (labelled –2 and 4), and the ‘+l peaks’ of these ‘slippage peaks’. For this marker, the normal peaks and the +l peaks have a similar height. That means that, depending on the samples, the Genotyper software will choose one peak or the other for the allele, leading to errors in the genetic data.

Published
1996
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