Biased sinusoidal field gel electrophoresis for the separation of large DNA.

Abstract

In agarose gel electrophoresis, in a steady, continuous field, it is well known that the mobility mu, 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, is weak; entangled but unstretched regime II for intermediate-size L-DNAs (of M < 20 kbp), where mu, 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 regimen boundaries can be altered by adjusting the gel concentration Cgel 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 (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.(ABSTRACT TRUNCATED AT 250 WORDS)