Separation and size determination of circular and linear single-stranded DNAs by alkaline agarose gel electrophoresis

The electrophoretic mobility of single-stranded DNA (ssDNA) through agarose gels depends on size (number of bases; molecular weight), topology (linear or circular), and conformation (secondary and tertiary folding through base pairing and stacking), as well as on gel concentration and field strength. Under alkaline conditions that disrupt secondary and tertiary structures, the linear and circular forms of a given single-stranded DNA can be easily resolved. The mobility ratios, mulinear/mucircular, depend strongly on gel concentration and less strongly on DNA size. Protocols have been developed to determine conveniently the size within 2% and the fractions of circular and linear forms for ssDNA sizes on the order of 10 kb.