BIASED SINUSOIDAL FIELD GEL-ELECTROPHORESIS FOR LARGE DNA SEPARATION

For size-dependent separation of DNAs, we propose a new electrophoretic method, a 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). The BSFGE was tested on double-stranded DNAs with molecular weight (M) ranging from 200 basepairs (bp) to 2 Mbp in agarose gel of concentration (C(gel)) from 0.25 to 1.5 wt %. From the M dependence of the electrophoretic mobility (mu-s) under a low steady field, the features were classified into three regimes that are similar to Slater-Noolandi's regimes: the Ogston regime, I; the entanglement (without stretching) regime, II; and the entanglement (with stretching) regime, III. The BSFGE results exhibited three features depending on the conditions: Under a high bias with E(b) greater-than-or-equal E(s) (greater-than-or-equal-to 5.0 V cm-1) and varying f, a significant maximum in mu-was observed on all DNAs at an fM independent of M but dependent on C(gel) as fM proportional-to C(gel)7. Under an intermediate bias with E(b) less-than-or-equal-to 5.0 V cm-1 less-than-or-equal-to E(s); mu, starting from mu-s at high f, increases with decreasing f and reaches a plateau (mu-0) at low frequencies for DNAs of M smaller than 10 kbp; the transition frequency (f(T)) was nearly independent of M, when M was large. The ratio-mu-0-mu-s-1 did not depend on M in regimes I and III but increased with M in regime II. Under still lower bias, mu for DNAs with M > 20 kbp, again starting from mu-s at high f, reached a pronounced minimum at a frequency (f(p), pin-down frequency) specific to M and C(gel) in such a way that f(p) proportional-to C(gel)-1 M-1, before reaching the low-f plateau (mu-0).