title: Fenton fragmentation for faster electrophoretic on chip purification of amplifiable genomic DNA.
abstract: With a rapid and simple actuation protocol electrophoretic nucleic acid extraction is easy automatable, requires no moving parts, is easy to miniaturize and furthermore possesses a size dependent cut-off filter adjustable by the pore size of the hydrogel. However electrophoretic nucleic acid extraction from bacteria has so far been applied mainly for short RNA targets. One of the reasons is that electrophoretic processing of unfragmented genomic DNA strands is time-consuming, because of the length. Here DNA fragmentation would accelerate extraction and isolation. We introduce on-chip lysis and non-enzymatic DNA cleavage directly followed by a purifying step for receiving amplifiable DNA fragments from bacteria in less than 25 min. In contrast to restriction enzymes the Fenton reaction is known to cleave DNA without nucleotide specificity. The reaction mix contains iron(II) EDTA, sodium ascorbate, hydrogen peroxide and lysozyme. The degree of fragmentation can be adjusted by the concentration of reagents. The results enable electrophoretic extraction methods to unspecifically process long genomic DNA in a short time frame, e.g. for pathogen detection in a lab-on-a-chip format.
authors: Laboratory for Sensors, Department for Microsystem Engineering (IMTEK), Albert-Ludwigs-University Freiburg, Georges-Köhler-Allee 103, 79110 Freiburg, Germany. Electronic address: hakenberg@imtek.de.
authors: S
authors: S
authors: Hakenberg
authors: Laboratory for Sensors, Department for Microsystem Engineering (IMTEK), Albert-Ludwigs-University Freiburg, Georges-Köhler-Allee 103, 79110 Freiburg, Germany.
authors: M
authors: M
authors: Hügle
authors: Laboratory for Sensors, Department for Microsystem Engineering (IMTEK), Albert-Ludwigs-University Freiburg, Georges-Köhler-Allee 103, 79110 Freiburg, Germany.
authors: P
authors: P
authors: Meyer
authors: Laboratory for Sensors, Department for Microsystem Engineering (IMTEK), Albert-Ludwigs-University Freiburg, Georges-Köhler-Allee 103, 79110 Freiburg, Germany.
authors: O
authors: O
authors: Behrmann
authors: Laboratory for Sensors, Department for Microsystem Engineering (IMTEK), Albert-Ludwigs-University Freiburg, Georges-Köhler-Allee 103, 79110 Freiburg, Germany. Electronic address: dame@imtek.de.
authors: G
authors: G
authors: Dame
authors: Laboratory for Sensors, Department for Microsystem Engineering (IMTEK), Albert-Ludwigs-University Freiburg, Georges-Köhler-Allee 103, 79110 Freiburg, Germany. Electronic address: urban@imtek.de.
authors: G A
authors: GA
authors: Urban
mesh_terms: D015374:Biosensing Techniques; D053938:DNA Fragmentation; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D004586:Electrophoresis; D016680:Genome, Bacterial / Q000235:genetics; D006861:Hydrogen Peroxide / Q000737:chemistry; D007501:Iron / Q000737:chemistry; D056656:Lab-On-A-Chip Devices; D021141:Nucleic Acid Amplification Techniques
chemical_list: D004269:DNA, Bacterial; C045076:Fenton's reagent; D006861:Hydrogen Peroxide; D007501:Iron
keywords: DNA extraction; DNA fragmentation; Electrophoresis; Lab-on-a-chip; Lysis; Unspecific DNA cleavage