Biased sinusoidal field gel electrophoresis for the separation of large DNA

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Abstract
In agarose gel electrophoresis, in a steady, continuous field, it is well known that the mobility μs 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 μs is weak; entangled but unstretched regime II for intermediate‐size L‐DNAs (of M < 20 kbp), where μs σ M−1 so that efficient fractionation is possible; and entangled and stretched regime III for large L‐DNAs, where M dependence of μs is again weak. Although μs and the regime boundaries can be altered by adjusting the gel concentration Cgel and/or the field strength E, the features of the M dependence of μs are essentially unchanged. As to the effect of DNA topology on μs, we found that in dilute gels (Cgel < 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 (Cgel > 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 Es and frequency f superposed on a steady bias field of strength Eb. Striking results in BSFGE of low bias (Eb < Es) conditions were that (i) the effective mobility μ at low f (μo) is higher than that of μ∞ at high f, which is equal to the steady field value μs, and (ii) for large DNAs of M > 20 kbp the μ exhibits a minimum μp (pin‐down mobility) at a frequency fp (pin‐down frequency) specific to M, Cgel, and the field strengths in such a way that fp σ M−1 Cgel −1Eb. Esα with α changing from 0 to 2 ∼ 3 at a value of Es dependent on Eb. The μp values appear to fall on the extrapolated portion of the regime II log(μs) 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.