Observation of Individual DNA Molecules Undergoing Gel Electrophoresis

Smith, Steven B.; Aldridge, Paul K.; Callis, James B.

doi:10.1126/science.2911733

Cited by 311 publications

(187 citation statements)

References 11 publications

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“…Since this oscillatory behavior appears as a steady state [8], the averaged conformation which the reptation model predicts is not stable. Also experimentally, since fluorescent microscopy has been invented and improved, evidences showing this instability have increased as well, and now details of DNA motion itself are of current interest in the study of gel electrophoresis [9,10,11,12]. Among them the inch-worm like motion may be the most typical example which is observed for large DNA [13].…”

Section: Introductionmentioning

confidence: 99%

Bond fluctuation method for a polymer undergoing gel electrophoresis

Azuma

Takayama

1999

Phys. Rev. E

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We present a new computational methodology for the investigation of gel electrophoresis of polyelectrolytes. We have developed the method initially to incorporate sliding motion of tight parts of a polymer pulled by an electric field into the bond fluctuation method (BFM). Such motion due to tensile force over distances much larger than the persistent length is realized by non-local movement of a slack monomer at an either end of the tight part. The latter movement is introduced stochastically. This new BFM overcomes the well-known difficulty in the conventional BFM that polymers are trapped by gel fibers in relatively large fields. At the same time it also reproduces properly equilibrium properties of a polymer in a vanishing filed limit. The new BFM thus turns out an efficient computational method to study gel electrophoresis in a wide range of the electric field strength.

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Section: Introductionmentioning

confidence: 99%

Bond fluctuation method for a polymer undergoing gel electrophoresis

Azuma

Takayama

1999

Phys. Rev. E

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“…Direct observations in the light microscope indicate that DNA moves through agarose by oscillating among a complex repertoire of configurations (18)(19)(20). In a continuous field, DNA molecules fluctuate between extended and tangled configurations, but with a general orientation in line with the applied field (Fig.…”

Section: Introductionmentioning

confidence: 99%

Bag model for DNA migration during pulsed-field electrophoresis.

Chu

1991

Proc. Natl. Acad. Sci. U.S.A.

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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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“…The slinky theory also predicts that the molecular motion of the DNA molecule undergoing electrophoresis would be quite different from the motion predicted by tube theories (Deutsch, 1988). Experimental observations of stained DNA molecules undergoing CF electrophoresis (Schwartz and Koval, 1989;Smith et al, 1989), conducted after Deutsch proposed his model, strongly support the slinky theory.…”

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confidence: 52%

“…Mathematical modelling Slater et al, 1987;Viovy, 1987;Viovy, 1989;Willis et al, 1988;Deutsch, 1987Deutsch, , 1988Deutsch and Madden, 1989), observation of single DNA molecules in agarose gels Schwartz and Koval, 1989), linear and electric dichroism studies (Holzwarth et al, 1987;Akerman et al, 1989;Porsche, 1989), electric birifringence measurements (Stellwagen, 1985;Stellwagen andStellwagen, 1989a, 1989b), scanning tunnel microscopy , observation of the movement of fluorescence patterns after photobieaching (Chu et al, 1989), and computer simulations (Lalande et al, 1987;Batoulis et al, 1989;Deutsch, 1988;Smith et al, 1989) (Heller and Pohl, 1989).…”

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Pulsed-field gel electrophoresis studies of Rhizobiaceae

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Observation of Individual DNA Molecules Undergoing Gel Electrophoresis

Cited by 311 publications

References 11 publications

Bond fluctuation method for a polymer undergoing gel electrophoresis

Bond fluctuation method for a polymer undergoing gel electrophoresis

Bag model for DNA migration during pulsed-field electrophoresis.

Pulsed-field gel electrophoresis studies of Rhizobiaceae

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