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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 with either biotin or digoxigenin. We obtained a relatively high yield of gel-purified DNA (∼500 ng/100 µl of PCR reaction). The final construct in this PCR-based method contains only one labelled strand in contrast to the traditional construct in which both strands of the DNA are labelled. Nonetheless, we achieved a high yield (84%) of torsionally constrained DNA when measured using an optical-trap-based DNA-overstretching assay. This protocol significantly simplifies the application and adoption of torsionally constrained assays to a wide range of single-molecule systems.

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