Bag model for DNA migration during pulsed-field electrophoresis.

Abstract

A model for pulsed-field electrophoresis was developed by picturing large DNA as a deformable "bag" that (i) moves with limiting mobility in a continuous electric field,(ii) adopts an orientation aligned with the field direction, and (ii) reorients after a change in field direction in a sizedependent manner. The model correctly predicted the resolution of large DNA in a pulsed field inoudin the surprising phenomena of mobility inversion, lateral band spreading, and improved resolution for obtuse angles. A simple parametrization agreed with observations of two completely different aspects ofDNA behavior: bulk mobility as measured during gel electrophoresis and molecular reorientation as measured by linear dichroism. The model also provides quantitative guidelines for setting experimental parameters in pulsed-field electrophoresis experiments.Gel electrophoresis in a continuous electric field does not resolve DNA molecules larger than =50 kilobase pairs (kbp) (1). Large DNA moves through the gel matrix but with a velocity that is independent of size. However, if the electric field is reoriented periodically, DNA up to several megabase pairs (Mbp) may be resolved (2-4). Because of widespread interest in separating large DNA molecules, there has been a growing effort to understand the physical basis ofpulsed-field electrophoresis (5). An early model assumes that DNA moves through the gel in a fully extended conformation: when the field is reoriented by an angle >90°, the DNA begins moving in the new direction led by what was formerly its back end (6). This simple model successfully predicts the separation of large DNA in a pulsed electric field. However, contrary to what is observed, it also predicts that the upper size limit of resolution is accompanied by zero DNA mobility. This model will not be considered further, since the goal here is to build a model that is quantitatively correct.A second model pictures the motion of the DNA as a "reptating" chain that crawls snakelike through a tube in the gel in response to the electric field. As the DNA moves forward, its leading end creates additional segments in the tube. Each step forward is in a random direction but with a bias in favor of the electric field direction. This relaxation from the rigid assumption of a fully extended conformation allows more complex DNA behavior. The reptation model successfully predicts that continuous fields will fail and pulsed fields will succeed in separating large DNA (7-9) and that in a pulsed field the size limit of separation increases proportionally to pulse time and is associated with a nonzero mobility (10). It also predicts two unexpected phenomena. First, under some conditions, transversely pulsed fields should cause larger DNA molecules to migrate faster than smaller species ("mobility inversion"). This has been experimentally observed in electrophoresis with periodic inversion of the electric field (3), but the reptation model predicts that it should also occur in transversely pulsed fields (10). Second, similar co...

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“…However, the accurate analysis of the profile of the continuous fragment distribution is not trivial Kraxenberger et al, 1994;Cedervall et al, 1995). In addition, PFGE could be complicated by paradoxical migration patterns (Carle et al, 1986;Chu, 1991;Löbrich et al, 1993). Therefore, PFGE and constant-field gel electrophoresis (CFGE) are preferably used to quantify only the fraction of DNA fragments released (FDR) from the bulk DNA (Blöcher et al, 1989;Stamato and Denko, 1990;Iliakis et al, 1991a, b).…”

Nonmonotonous variation of DNA angular separation during asymmetric pulsed field electrophoresis

“…The combination of DNA deformation and migration mechanisms results in the frequency response of the mobility, i.e. a decline, plateau, and a rise in the μ – f curve in PFGE . The same deformations and mechanisms give the frequency response observed in APFE, i.e.…”

“…However, the nonmonotonic behavior of θ with f is rather complicated . In addition to pulse time, another critical time scale in any pulsed field electrophoresis scheme either in gel or microfabricated array is the reorientation time of DNA, t or . This reorientation time has been defined as the time it takes DNA to completely align itself to the new direction of the switched electric field .…”

Electrophoretic Karyotyping of Yeasts

“…Since the theory of pulsed-field gel electrophoresis (PFGE) is rather complex, a short, simplified explanation will be given. For a detailed description of PFGE techniques and theory, see Gemmill (1991) and Chu (1991). The DNA is electrophoresed through an agarose gel, the driving forces are electrical fields which are turned on alternatively.…”