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Biased sinusoidal field gel electrophoresis for the separation of large DNA
Author(s) -
Kotaka Tadao,
Adachi Shiro,
Shikata Toshiyuki
Publication year - 1993
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150140154
Subject(s) - pulsed field gel electrophoresis , separation (statistics) , chromatography , electrophoresis , gel electrophoresis , field (mathematics) , dna , separation method , chemistry , computer science , biochemistry , mathematics , gene , machine learning , genotype , pure mathematics
In agarose gel electrophoresis, in a steady, continuous field, it is well known that the mobility μ 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 μ s is weak; entangled but unstretched regime II for intermediate‐size L‐DNAs (of M < 20 kbp), where μ s σ M −1 so that efficient fractionation is possible; and entangled and stretched regime III for large L‐DNAs, where M dependence of μ s is again weak. Although μ 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 μ s are essentially unchanged. As to the effect of DNA topology on μ 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 μ at low f (μ o ) is higher than that of μ ∞ at high f , which is equal to the steady field value μ s , and (ii) for large DNAs of M > 20 kbp the μ exhibits a minimum μ 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 σ M −1 C gel −1 E b . E s α with α changing from 0 to 2 ∼ 3 at a value of E s dependent on E b . The μ p values appear to fall on the extrapolated portion of the regime II log(μ 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.