Molecular Motors

Damian J. delToro, Douglas E. Smith, delToro, Damian J., Smith, Douglas E.

The unwinding of double-stranded DNA is a frequently occurring event during the cellular processes of DNA replication, repair, and transcription. To help further investigate properties of this fundamental process as well as to study proteins acting on unzipped DNA at the single molecule level, we describe a novel method for efficient preparation of long DNA constructs (arbitrary sequences of many kilobasepairs (kbp) in length) that can be forcibly unzipped and manipulated with optical tweezers or other single-molecule manipulation techniques. This method utilizes PCR, a nicking endonuclease, and strand displacement synthesis by the Klenow fragment of DNA polymerase I to introduce labeled nucleotides at appropriate positions to facilitate unzipping of the DNA by application of force. We also describe various optical tweezers measurement modes for measuring DNA unzipping and rezipping. These methods have applications to studying helicases and DNA binding proteins.