Direct and quantitative electrophoretic detection of covalently closed DNA circles formed by in vitro ligation.

The typical products of enzymatic circularization of DNA, using DNA ligase or recombinase, are covalently closed and mostly relaxed DNA circles. Because they are difficult to analyze on conventional gels, they are often converted to nicked circles prior to electrophoresis. Herein, we present a sensitive and quantitative procedure for directly analyzing ligated closed circle DNA on agarose gels without additional treatments. Specifically, inclusion of GelStar dye in the gel allowed detection of ligated closed circle DNAs, which were likely super-twisted by being intercalated by GelStar, as discrete bands with good separation from linear DNA of the same sizes.