Gel electrophoresis of end-labeled DNA. II. Dynamics and detrapping in pulsed fields.
A theory for field-inversion gel electrophoresis of a flexible polyelectrolyte bearing an uncharged bulky label at one end is described, and the evolution of the mobility with chain length, field strength, friction of the label, and the duration of the forward and reverse pulses is predicted. A new critical size, Ndetrap, is introduced, and its value calculated. It increases roughly linearly with the duration of the reverse pulses. Chains smaller than Ndetrap are detrapped by reverse pulses, and may have a high mobility, whereas chains larger than Ndetrap are not trapped, and have a very small mobility. This leads to an increase of the mobility (as compared with constant field) in a given range of sizes, and to a strong selectivity around Ndetrap. Depending on the parameters, numerous other effects, including a secondary mobility plateau and band inversion, may appear. The corresponding regimes are predicted and discussed. All predictions are qualitatively consistent with available experimental data. We use them to suggest efficient conditions for the development of pulsed-field trapping electrophoresis, a possible tool for improved DNA sequencing. In particular, we recommend using a ramping of pulse times, with a constant ratio of forward to reverse time in the range 3 to 5.
Défontaines
A D
AD
Laboratoire de Physicochimie théorique, ESPCI, Paris, France.
Viovy
J L
JL
D004247:DNA / Q000032:analysis*; D004574:Electromagnetic Fields; D016521:Electrophoresis, Gel, Pulsed-Field; D015233:Models, Statistical
D004247:DNA