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 ∼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. Graha Mandal Horoscope Software on this page. An alternative stronger attachment method using an amino–carboxyl linkage, which allows for reliable DNA overstretching, is also described.

INTRODUCTION Protein–DNA interactions play a critical role in the molecular biology of all organisms. For example, the ∼3.3 billion base pairs human genome is estimated to code for at least several thousand DNA-binding proteins, including transcription factors, nucleases, repair proteins, topoisomerases, structural proteins, and DNA and RNA polymerases. A wide variety of methods exist for studying protein–DNA interactions, including DNase footprinting, sucrose gradient sedimentation, gel mobility shifts, fluorescence spectroscopy, imaging by electron microscopy and X-ray crystallography. Over the last decade another approach involving mechanical manipulation of single DNA molecules has been developed. Manipulation of DNA by optical tweezers was pioneered by Chu and co-workers, and extended by Bustamante and co-workers ( – ). This method has since been applied to study many fundamental biochemical processes, including transcription, replication, chromatin unraveling, viral DNA packaging and helicase translocation ( – ). Because protein–DNA interactions are vital to all organisms and these interactions are often sequence dependent, it is desirable to have a general method for manipulating DNA sequences from any organism.