Dynamics of DNA Molecules in Gel Studied by Fluorescence Microscopy

Kantor, Ronald; Guo, Xuan-Hui; Huff, Edward J.; Schwartz, David C.

doi:10.1006/bbrc.1999.0592

Cited by 13 publications

(12 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…8,9 Much less theoretical or computational work is available on the dynamics of confined chains, either in equilibrium or flow, in spite of their practical importance. [10][11][12][13][14][15] Qualitatively, one might envision two effects of confinement on the dynamics of a dissolved chain. First, the change in equilibrium conformation brought on by confinement may change the molecular motion ͑see Fig.…”

Section: Introductionmentioning

confidence: 99%

Effect of confinement on DNA dynamics in microfluidic devices

Jendrejack

Schwartz

Graham

et al. 2003

The Journal of Chemical Physics

Self Cite

166

193

View full text Add to dashboard Cite

The dynamics of dissolved long-chain macromolecules are different in highly confined environments than in bulk solution. A computational method is presented here for detailed prediction of these dynamics, and applied to the behavior of ϳ1-100 m DNA in micron-scale channels. The method is comprised of a self-consistent coarse-grained Langevin description of the polymer dynamics and a numerical solution of the flow generated by the motion of polymer segments. Diffusivity and longest relaxation time show a broad crossover from free-solution to confined behavior centered about the point HϷ10S b , where H is the channel width and S b is the free-solution chain radius of gyration. In large channels, the diffusivity is similar to that of a sphere diffusing along the centerline of a pore. For highly confined chains (H/S b Ӷ1), Rouse-type molecular weight scaling is observed for both translational diffusivity and longest relaxation time. In the highly confined region, the scaling of equilibrium length and relaxation time with H/S b are in good agreement with scaling theories. In agreement with the results of Harden and Doi ͓J. Phys. Chem. 96, 4046 ͑1992͔͒, we find that the diffusivity of highly confined chains does not follow the scaling relation predicted by Brochard and de Gennes ͓J. Chem. Phys. 67, 52 ͑1977͔͒; that relationship does not account for the interaction between chain and wall.

show abstract

Section: Introductionmentioning

confidence: 99%

Effect of confinement on DNA dynamics in microfluidic devices

Jendrejack

Schwartz

Graham

et al. 2003

The Journal of Chemical Physics

Self Cite

166

193

View full text Add to dashboard Cite

show abstract

“…Understanding the transition from transient entanglement coupling to reptation and how it correlates with polymer properties can aid in the development of polymer solutions tailored for specific DNA separation requirements, thereby maximizing performance and minimizing separation time. In more recent work, the Barron group studied these three regimes and the transitions between them via single-molecule videomicroscopy of fluorescently labeled λ-DNA fragments utilizing three common separation matrices [45, 46]: linear polyacrylamide (LPA), hydroxyethylcellulose (HEC), and poly(ethylene oxide) (PEO), correlating the occurrence of TEC and reptation events with polymer molar mass and concentration [40]. To accomplish this, a minimum of two 30-second videos were recorded and an average of 30-40 separate DNA molecules' migration mechanisms were tabulated as they electrophoresed through a given polymer solution.…”

Section: Stochastic Single-molecule Videomicroscopy Methods To Measmentioning

confidence: 99%

DNA migration mechanism analyses for applications in capillary and microchip electrophoresis

et al. 2009

View full text Add to dashboard Cite

In 2009, electrophoretically driven DNA separations in slab gels and capillaries have the sepia tones of an old-fashioned technology in the eyes of many, even while they remain ubiquitously used, fill a unique niche, and arguably have yet to reach their full potential. For comic relief, what is old becomes new again: agarose slab gel separations are used to prepare DNA samples for "next-gen" sequencing platforms (e.g., the Illumina and 454 machines)-dsDNA molecules within a certain size range are "cut out" of a gel and recovered for subsequent "massively parallel" pyrosequencing. In this review, we give a Barron lab perspective on how our comprehension of DNA migration mechanisms in electrophoresis has evolved, since the first reports of DNA separations by CE (∼1989) until now, 20 years later. Fused silica capillaries, and borosilicate glass and plastic microchips, quietly offer increasing capacities for fast (and even "ultra-fast"), efficient DNA separations. While the channel-by-channel scaling of both old and new electrophoresis platforms provides key flexibility, it requires each unique DNA sample to be prepared in its own micro-or nanovolume. This Achille's heel of electrophoresis technologies left an opening through which pooled-sample, next-gen DNA sequencing technologies rushed. We shall see, over time, whether sharpening understanding of transitions in DNA migration modes in crosslinked gels, nanogel solutions, and uncrosslinked polymer solutions will allow electrophoretic DNA analysis technologies to flower again. Microchannel electrophoresis, after a quiet period of metamorphosis, may emerge sleeker and more powerful, to claim its own important niche applications.

show abstract

“…The possibility exists that this feature of the pulse sequence could play a role in the separation if it becomes comparable to the DNA relaxation time. Experimental studies of electrophoretically stretched DNA relaxation in agarose gels suggest that the timescale associated with the most rapid relaxation mode scales approximately as N 1.5 (where N is the DNA fragment length in base pairs), consistent with Zimm‐like behavior 33, 34. But the absolute value of the relaxation time is more challenging to predict quantitatively because of sensitivity to experimental details including the concentration and composition of the gel and buffer.…”

Section: Comparison Between Size Dependence Of Dna Mobility Observed mentioning

confidence: 96%

“…Kantor et al . obtained a relationship for the relaxation time τ (s)=7.6×10 −7 N 1.45 based on fluorescence imaging of individual molecules in the 245–980 kb range 33, while Sturm and Weill obtained τ (s)=4×10 −8 N 1.5 based on birefringence measurements of DNA in the 10–50 kb range 34. Taking a 30 kb fragment length as representative of the upper limit of sizes in our DNA ladder, these scalings predict relaxation times of about 2.4 and 0.21 s, respectively.…”

Section: Comparison Between Size Dependence Of Dna Mobility Observed mentioning

confidence: 99%

Investigating DNA migration in pulsed fields using a miniaturized FIGE system

Chen

Ugaz

2008

Electrophoresis

View full text Add to dashboard Cite

Pulsed field gel electrophoresis (PFGE) is a well-established technique for fractionation of DNA fragments ranging from kilobases to megabases in length. But many of these separations require an undesirable combination of long experiment times (often approaching tens of hours) and application of high voltages (often approaching tens of kV). Here we present a simple miniaturized field inversion gel electrophoresis (FIGE) apparatus capable of separating DNA fragments up to 32.5 kb in length within 3 hours using a modest applied potential of 20 V. The device is small enough to be imaged under a fluorescence microscope, permitting the migrating DNA bands to be observed during the course of the separation run. We use this capability to investigate how separation performance is affected by parameters including the ratio of forward and backward voltage, pulse time, and temperature. We also characterize the dependence of DNA mobility on fragment size N, and observe a scaling in the vicinity of N −0.5 over the size range investigated. The high speed, low power consumption, and simple design of this system may help enable future studies of DNA migration in PFGE to be performed quickly and inexpensively.Keywords pulsed field gel electrophoresis; field inversion gel electrophoresis; DNA As DNA fragment lengths approach tens of kilobases, a point is reached where individual molecules become much larger than the average pore size of the gel matrix. In order to sustain electrophoretic migration in this regime, the DNA molecules must adopt extended conformations preferentially aligned in the electric field direction. But this mode of migration is accompanied by a drastic reduction (and ultimately saturation) in the size dependence of electrophoretic mobility, making separation of long DNA fragments under continuous electric fields extremely challenging if not impossible. One way to overcome these limitations involves introducing a dynamic reorientation process by periodically changing the direction and magnitude of the applied electric field. When the electric field direction is switched, smaller fragments are able to reorient more quickly than larger molecules and thus migrate faster. The size dependent nature of this reorientation process restores the ability to distinguish different sized fragments based on their electrophoretic mobility. These pulsed field gel electrophoresis (PFGE) techniques have greatly extended the range of DNA fragment sizes that can be separated, even up to tens of Mb [1,2]. PFGE methods are important in a variety of applications including analysis of microbial genomes [3][4][5] [7][8][9][10][11]. But despite the instrumental role of PFGE techniques in broadening the range of DNA fragment sizes that can be effectively separated, some important drawbacks remain. First, PFGE can be extremely time consuming, with run times generally ranging in the tens of hours. A major reason for this is that the extent to which the electric field can be increased to drive faster DNA migration is limited by the ...

show abstract

Dynamics of DNA Molecules in Gel Studied by Fluorescence Microscopy

Cited by 13 publications

References 34 publications

Effect of confinement on DNA dynamics in microfluidic devices

Effect of confinement on DNA dynamics in microfluidic devices

DNA migration mechanism analyses for applications in capillary and microchip electrophoresis

Investigating DNA migration in pulsed fields using a miniaturized FIGE system

Contact Info

Product

Resources

About