title: A general method for manipulating DNA sequences from any organism with optical tweezers.
abstract: Mechanical manipulation of single DNA molecules can provide novel information about DNA properties and protein-DNA interactions. Here we describe and characterize a useful method for manipulating desired DNA sequences from any organism with optical tweezers. Molecules are produced from either genomic or cloned DNA by PCR using labeled primers and are tethered between two optically trapped microspheres. We demonstrate that human, insect, plant, bacterial and viral sequences ranging from approximately 10 to 40 kilobasepairs can be manipulated. Force-extension measurements show that these constructs exhibit uniform elastic properties in accord with the expected contour lengths for the targeted sequences. Detailed protocols for preparing and manipulating these molecules are presented, and tethering efficiency is characterized as a function of DNA concentration, ionic strength and pH. Attachment strength is characterized by measuring the unbinding time as a function of applied force. An alternative stronger attachment method using an amino-carboxyl linkage, which allows for reliable DNA overstretching, is also described.
authors.affiliation: Department of Physics, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0379, USA.
authors.forename: Derek N
authors.initials: DN
authors.lastname: Fuller
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Gemmen
authors.forename: John Peter
authors.initials: JP
authors.lastname: Rickgauer
authors.forename: Aurelie
authors.initials: A
authors.lastname: Dupont
authors.forename: Rachel
authors.initials: R
authors.lastname: Millin
authors.forename: Pierre
authors.initials: P
authors.lastname: Recouvreux
authors.forename: Douglas E
authors.initials: DE
authors.lastname: Smith
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000302:isolation & purification; D004548:Elasticity; D005821:Genetic Techniques; D006801:Humans; D007834:Lasers; D046210:Microfluidic Analytical Techniques; D008863:Microspheres; D016133:Polymerase Chain Reaction
chemical_list: D004247:DNA