Periodic Motion of Large DNA Molecules during Steady Field Gel Electrophoresis

Oana, Hidehiro; Masubuchi, Yuichi; Matsumoto, Mitsuhiro; Doi, Masao; Matsuzawa, Yukiko; Yoshikawa, Kenichi

doi:10.1021/ma00099a019

Cited by 60 publications

(86 citation statements)

References 6 publications

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“…A chain moving in a relatively collapsed form (a) is trapped by a certain obstacle (b), deforms to a V-or U-shaped conformation (c,d), slides off the obstacle (e), and then tends to form a collapsed conformation again (f). In contrast to experimental observation [13], however, quasi-periodical alternation between contracted and extended conformations has not yet been observed, or periods between the two conformations are rather random.…”

Section: Resultscontrasting

confidence: 94%

“…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%

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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: Resultscontrasting

confidence: 94%

Section: Introductionmentioning

confidence: 99%

Bond fluctuation method for a polymer undergoing gel electrophoresis

Azuma

Takayama

1999

Phys. Rev. E

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“…They found that YOYO-stained T2 DNA molecules in 1% agarose gel at 8 V/cm in average spend 68% of the oscillation time in the U-formation/deformation step, 22 Yo in the I-contraction step, and 10% as compressed conformations. These data are consistent with the observation that the velocity of the tail end of the DNA molecule during the I-contraction step is about four times higher than the velocity of the front part (which is constant during the cycle), i.e., the molecule spends, on average, three times more time in the U-formation/deformation step than in the I-contraction step [22]. According to Eq.…”

Section: Simulationssupporting

confidence: 90%

Simulations of the overshoot in the build‐up of orientation of long DNA during gel electropohoresis based on a distribution of oscillation times

Carlsson

Larsson

1996

Electrophoresis

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The periodic extension-contraction motion observed for long DNA molecules undergoing agarose gel electrophoresis in a constant field is believed to be important for the separation mechanism in pulsed field gel electrophoresis. These oscillations give rise to an overshoot and an undershoot in the ensemble orientation of DNA in the beginning of a field pulse, when the molecules oscillate coherently. After approximately one oscillation cycle, the coherence between the molecules is lost, and a constant, cycle-averaged orientation is reached. In this paper we simulate this build-up of the ensemble orientation of DNA by using a distribution of oscillation times (the time between two consecutive compressed conformations) determined by fluorescence microscopy for YOYO-stained DNA. Six different orientation profiles, describing the orientation during one oscillation cycle, were used. The simulated orientation responses are compared with an orientation response measured by linear dichroism (LD) under the same experimental conditions as in the microscopy study. We found that the choice of orientation profile during the oscillation is important. Best agreement between the simulated and the experimental orientation response was obtained for an orientation profile based on a theoretical model by Schurr and Smith (Biopolymers 1990, 29, 1161-1165). The influence of the distribution of oscillation times and its standard deviation on the orientation response was also investigated. Furthermore, simulations at different field strengths and DNA sizes were performed and found to agree quite well with the experimentally obtained LD data.

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“…By optimally shaping the field pulses, the molecular reorientation time can be cut in half (compared with constant-height pulses), reducing proportionally the separation time and still avoiding trapping. Assume that the pulse has been reversed for a time t and that the arms extend (from zero length) at the rate E, where is the mobility of the DNA (4,29). The force builds in time according to F ϭ eff E 2 t until the molecular contour length, L, is reached, and then F Ͻ eff EL͞2.…”

Section: Discussionmentioning

confidence: 99%

Trapping of megabase-sized DNA molecules during agarose gel electrophoresis

Gurrieri¹,

Smith²,

Bustamante³

1999

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

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Megabase DNA molecules become trapped in agarose gels during electrophoresis if the electric field exceeds a few volts per cm. Fluorescence microscopy reveals that these molecules invariably arrest in U-shaped conformations. The field-vs.-size dependence for trapping indicates that a critical molecular tension is required for trapping. The size of unligated -ladders, sheared during gel electrophoresis at a given field, coincides with the size of molecules trapped at that field, suggesting that both processes occur through nick melting near the vertex of the U-shape. Consistently, molecules nicked by exposure to UV radiation trap more readily than unexposed ones. The critical trapping tension at the vertex is estimated to be 15 pN, a force sufficient to melt nicks bent around gel fibers, and, according to our model, trap a molecule. Strategies to reduce molecular tension and avoid trapping are discussed.Gel electrophoresis is the method of choice for the size fractionation of DNA in analytical biochemistry. Steady-field electrophoresis is commonly used to separate molecules from a few bp to about 20 kbp, while pulsed-field gel electrophoresis (PFGE) methods (1-3) are required to separate molecules beyond this size range, up to 10 megabase pairs (Mbp). In PFGE, the electric field is periodically alternated in two directions and DNA separation depends on the way the molecules reorient through the gel in response to the changing electric field (4, 5). Unfortunately, molecules longer than 1-2 Mbp can become permanently immobilized or trapped after traveling various distances through the gel, leading to band smearing (6). Trapping occurs in both steady-field and pulsedfield experiments if the electric field is higher than some critical value, E crit (7). The value of E crit falls as the DNA size is increased. Lowering the field below 2 V͞cm prevents trapping (6) but at the expense of very long electrophoretic runs (4). E crit appears to be higher for PFGE than for steady-field electrophoresis. Trapping in PFGE has been reduced, and sharper bands obtained by interrupting the long electric-field pulses with short high-voltage spikes in the reverse direction (7). These protocols notwithstanding, DNA trapping still remains a practical barrier to high-resolution PFGE above 10 Mbp. Understanding and preventing molecular trapping is essential to overcoming the current size limit of DNA separation and may lead to sharper resolution and reduced running times.Several mechanisms of DNA trapping have been proposed. Olson (6) suggested that very large molecules pile up against pores in a compressed state. Turmel and coworkers (7) suggest such pileups may occur in agarose cul-de-sacs because of locally high gel concentrations. Alternately, multiple ''hernias'' or ''impaled spirals'' (large DNA knots) could be the cause (7).Deutsch (8) has proposed that the molecules may bend around gel fibers with the two arms extending in the direction of the electric field, forming U-shapes. Because of local corrugation in both the DNA an...

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Periodic Motion of Large DNA Molecules during Steady Field Gel Electrophoresis

Cited by 60 publications

References 6 publications

Bond fluctuation method for a polymer undergoing gel electrophoresis

Bond fluctuation method for a polymer undergoing gel electrophoresis

Simulations of the overshoot in the build‐up of orientation of long DNA during gel electropohoresis based on a distribution of oscillation times

Trapping of megabase-sized DNA molecules during agarose gel electrophoresis

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