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

In agarose gel electrophoresis, in a steady, continuous field, it is w ell known that the mobility mu(s) versus size M relation for linear DN As (L-DNAs) can be divided into three regimes: Ogston regime I for sma ll DNAs, where M dependence of mu(s) is weak; entangled but unstretche d 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 a nd 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 altere d 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 g els (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 g els (C(gel) > 1.5 wt%) C-DNAs migrate much slower than L-DNAs.To facil itate separation of large DNAs in the regime III range, we proposed bi ased sinusoidal field gel electrophoresis (BSFGE), which utilizes a si nusoidal 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 c hanging 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 interprete d in terms of the current dynamical models of DNA gel electrophoresis and also with the results of direct observation by fluorescence micros copy on migrating T4dC DNA of 166 kbp in a steady field and under seve ral BSFGE conditions.