title: A single molecule assay for measuring site-specific DNA cleavage.
abstract: Sequence-specific DNA cleavage is a key step in a number of genomic transactions. Here, we report a single-molecule technique that allows the simultaneous measurement of hundreds of DNAs, thereby collecting significant statistics in a single experiment. Microbeads are tethered with single DNA molecules in a microfluidic channel. After the DNA cleavage reaction is initiated, the time of cleavage of each DNA is recorded using video microscopy. We demonstrate the utility of our method by measuring the cleavage kinetics of NdeI, a type II restriction endonuclease.
authors.affiliation: Department of Chemistry and Physics, Emmanuel College, Boston, MA 02115, USA.
authors.forename: Stefano
authors.initials: S
authors.lastname: Gambino
authors.affiliation: Department of Chemistry and Physics, Emmanuel College, Boston, MA 02115, USA.
authors.forename: Briana
authors.initials: B
authors.lastname: Mousley
authors.affiliation: Department of Chemistry and Physics, Emmanuel College, Boston, MA 02115, USA.
authors.forename: Lindsay
authors.initials: L
authors.lastname: Cathcart
authors.affiliation: Department of Chemistry and Physics, Emmanuel College, Boston, MA 02115, USA.
authors.forename: Janelle
authors.initials: J
authors.lastname: Winship
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Loparo
authors.affiliation: Department of Chemistry and Physics, Emmanuel College, Boston, MA 02115, USA. Electronic address: priceal@emmanuel.edu.
authors.forename: Allen C
authors.initials: AC
authors.lastname: Price
mesh_terms: D002623:Chemistry Techniques, Analytical / Q000379:methods*; D004247:DNA / Q000378:metabolism*; D053837:DNA Cleavage; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D007700:Kinetics; D046210:Microfluidic Analytical Techniques; D018715:Microscopy, Video
chemical_list: D004247:DNA; C037106:endodeoxyribonuclease NdeI; D015252:Deoxyribonucleases, Type II Site-Specific
keywords: DNA; DNA cleavage; Restriction endonuclease; Single molecule