Torsionally constrained DNA for single-molecule assays: an efficient, ligation-free method.

ABSTRACT: Controlled twisting of individual, double-stranded DNA molecules provides a unique method to investigate the enzymes that alter DNA topology. Such twisting requires a single DNA molecule to be torsionally constrained. This constraint is achieved by anchoring the opposite ends of the DNA to two separate surfaces via multiple bonds. The traditional protocol for making such DNA involves a three-way ligation followed by gel purification, a laborious process that often leads to low yield both in the amount of DNA and the fraction of molecules that is torsionally constrained. We developed a simple ligation-free procedure for making torsionally constrained DNA via polymerase chain reaction (PCR). This PCR protocol used two 'megaprimers', 400-base-pair long double-stranded DNA that were labelled w

SUBMITTER: Paik DH 

PROVIDER: S-EPMC3799452 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature