BIASED SINUSOIDAL FIELD GEL-ELECTROPHORESIS FOR THE SEPARATION OF LARGE DNA

In agarose gel electrophoresis, in a steady, continuous field, it is well known that the mobility mu(s) versus size M relation for linear DNAs (L-DNAs) can be divided into three regimes: Ogston regime I for small DNAs, where M dependence of mu(s) is weak; entangled but unstretched regime II for intermediate-size L-DNAs (of M < 20 kbp), where mu(s) sigma M-1 so that efficient fractionation is possible; and entangled and stretched regime III for large, L-DNAs, where M dependence of mu(s) is again weak. Although mu(s) and the regime boundaries can be altered by adjusting the gel concentration C(gel) and/or the field strength E, the features of the M dependence of mu(s) are essentially unchanged. As to the effect of DNA topology on mu(s), we found that in dilute gels (C(gel) < 1.0 wt%) coiled, circular DNAs (C-DNAs) of 2-7 kbp size migrate faster than L-DNAs of comparable size, while in concentrated gels (C(gel) > 1.5 wt%) C-DNAs migrate much slower than L-DNAs.To facilitate separation of large DNAs in the regime III range, we proposed biased sinusoidal field gel electrophoresis (BSFGE), which utilizes a sinusoidal field of strength E(s) and frequency f superposed on a steady bias field of strength E(b). Striking results in BSFGE of low bias (E(b) < E(s)) conditions were that (i) the effective mobility mu at low, f(mu0) is higher than that of mu(infinity) at high f, which is equal to the steady field value mu(s), and (ii) for large DNAs of M > 20 kbp the mu exhibits a minimum mu(p) (pin-down mobility) at a frequency f(p) (pin-down frequency) specific to M, C(gel), and the field strengths in such a way that f(p) sigma M-1 C(gel)-1E(b).E(s)alpha with alpha changing from 0 to 2 approximately 3 at a value of E(s) dependent on E(b). The mu(p) values appear to fall on the extrapolated portion of the regime II log(mu(s)) versus log M curve. These results are interpreted in terms of the current dynamical models of DNA gel electrophoresis and also with the results of direct observation by fluorescence microscopy on migrating T4dC DNA of 166 kbp in a steady field and under several BSFGE conditions.