Controlling Electrophoretic Trapping of Circular DNA by Addition of Starch Preparations to Agarose Gels

Cole, Kenneth D.; Tellez, Carlos M.; Nguyen, Richard B.

doi:10.1385/abab:95:1:31

Cited by 4 publications

(4 citation statements)

References 12 publications

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“…A number of polymers were screened for enhancing circular DNA trapping [12]. Several polymers including polyethylene oxide (PEO), hydroxyethylcellulose (HEC), and dextran did not reduce the critical field strength required to trap the 13 kbp plasmid.…”

Section: Manipulation Of Concentration and Lengths Of Trapsmentioning

confidence: 99%

“…We have therefore studied the trapping process in detail, with the aim to use such a mechanistic understanding for the development of separation protocols for preparative scale purification of circular DNA forms. We here review results by ourselves and others [6][7][8][9][10][11][12][13] on the mechanism of trapping of relaxed circles, and related issues such as to what extent supercoiled DNA molecules can be trapped and how the capacity of the gel can be improved, i.e., how the density and volume of the traps can be increased. In the latter context some unpublished results on the effect of gel concentration have been included.…”

Section: Introductionmentioning

confidence: 99%

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Electrophoretic capture of circular DNA in gels

Åkerman

Cole

2002

Electrophoresis

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Results on electrophoretic capture of circular DNA in porous gels are reviewed. Processes which cause arrest of circular forms of DNA during electrophoresis can provide very efficient separation mechanism for the purification of plasmids and bacterial artificial chromosomes if the corresponding linear form is not trapped and therefore removed by the electric field. Two types of such topological traps have been proposed, impalement and lobster traps, and we here review the present experimental support for the existence of these two circle-specific mechanisms. Experiments designed to characterize the traps are discussed, regarding the concentration of the traps as well as their efficiency and capacity to trap both relaxed and supercoiled circular DNA. Studies of the dynamics of the capture process show that the average capture time is on the order of 10 s at 20 V/cm, by which time the circles have migrated several hundred micrometers and have passed hundreds of traps. We also review results on attempts to improve the capacity and efficiency of the trapping process by modification of the gels either by enzymatic treatment or by cogelation of neutral polymers.

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Section: Manipulation Of Concentration and Lengths Of Trapsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Electrophoretic capture of circular DNA in gels

Åkerman

Cole

2002

Electrophoresis

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“…The large open circles are readily trapped at low fields because their more relaxed structure is likely to encounter traps more frequently, and they have a low coefficient of diffusion that would hinder their escape. A number of gel factors will determine the trapping of circular DNA, including gel concentration [8], agarose molecular weight [9], and the choice of either agarose or polyacrylamide gel [10]. The characteristic trapping of open circular DNA on agarose gels can be used as a specific assay for DNA damage due to single-strand nicking of supercoiled DNA as a result of radiation or drugs [11].…”

Section: Introductionmentioning

confidence: 99%

Single-molecule measurements of trapped and migrating circular DNA during electrophoresis in agarose gels

2006

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The effect of agarose gel concentration and field strength on the electrophoretic trapping of open (relaxed) circular DNA was investigated using microscopic measurements of individual molecules stained with a fluorescent dye. Three open circles with sizes of 52.5, 115, and 220 kbp were trapped by the electric field (6 V/cm) and found to be predominately fixed and stretched at a single point in the gel. The length of the stretched circles did not significantly change with agarose concentration of the gels (mass fractions of 0.0025, 0.01, and 0.02). The relaxation kinetics of the trapped circles was also measured in the gels. The relaxation of the large open circles was found to be a slow process, taking several seconds. The velocity and average length of the 52.5 kbp open circles and 48.5 kbp linear DNA were measured during electrophoresis in the agarose gels. The velocity increased when the agarose concentrations were lowered, but the average length of the open-circle DNA (during electrophoresis) did not significantly change with agarose gel concentrations. The circles move through the gels by cycles of stretching and relaxation during electrophoresis. Linear dichroism was also used to investigate the trapping and alignment of the 52.5 kbp open circles. The results in this study provide information that can be used to improve electrophoretic separations of circular DNA, an important form of genetic material and commonly used to clone DNA.

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“…Our efforts have been to study the electrophoretic‐trapping process to better control and enhance it. Controlling and enhancing electrophoretic trapping would allow us to develop improved analytical and preparative methods for plasmid DNA [1, 2].…”

Section: Introductionmentioning

confidence: 99%

An apparatus for electrophoretic capture and recovery of circular DNA in thin layers

Cole

2003

Biotech and App Biochem

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An apparatus was designed for the electrophoretic capture and recovery of circular DNA in thin layers (membranes). Rapid separations were done by the use of a low-conductivity buffer and high electric field strengths. Two methods that specifically retain circular DNA in the membranes were demonstrated using the supercoiled and open circular forms of two plasmids with sizes of 4.4 and 13 kbp. Electrophoretic trapping (by an impalement mechanism) in agarose-gel-filled membranes used electric field strength to immobilize circular DNA in the membranes. The other method of capture utilized the greatly reduced electrophoretic mobility of circular DNA in membranes composed of agarose and the linear polymer hydroxyethylcellulose. The reduction in electrophoretic mobility was not dependent upon the electric field strength, distinguishing it from electrophoretic trapping. Trapping of circular DNA in the membranes followed by size analysis using agarose-gel electrophoresis could be used as a two-dimensional separation tool for the analysis of complex mixtures. Captured DNA was recovered by two methods: (i). centrifugation of membranes made with low-melting-point agarose resulted in a gel slurry that could be heated to release the DNA; (ii). electroelution of the membranes. Electroelution was done by using an electrode that was isolated from the DNA by a 'barrier' membrane. A non-trapping electric field was used to reverse the DNA out of the membrane into a small volume of buffer above the membrane.

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Controlling Electrophoretic Trapping of Circular DNA by Addition of Starch Preparations to Agarose Gels

Cited by 4 publications

References 12 publications

Electrophoretic capture of circular DNA in gels

Electrophoretic capture of circular DNA in gels

Single-molecule measurements of trapped and migrating circular DNA during electrophoresis in agarose gels

An apparatus for electrophoretic capture and recovery of circular DNA in thin layers

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