Information concerning the mechanism of electrophoretic DNA separation provided by quantitative video‐epifluorescence microscopy

Abstract

Changes in conformation, length, and mobility of individual DNA molecules during agarose gel electrophoresis were measured using video micrographs obtained by epifluorescence microscopy. Globular, V-shaped, and linear conformations of DNA are found. The mobility, upon transformation from the globular to the V-shaped conformation, decreases, suggesting a collision with a gel fiber. The duration of interaction between DNA and gel fiber is proportional to the length of DNA. Hypothetically, this proportionality underlies the size separation of DNA by agarose gel electrophoresis. DNA release from the gel fiber appears to involve the movement of the arms of the V-shaped molecule around the gel fiber. Concomitant with this movement is a length reduction the degree of which is constant for DNA of various lengths in a particular buffer milieu. The luminant densitometric profiles of DNA molecules in the V conformation show maxima at the ends and apex of the V. The unequal distribution of nucleotides along the DNA chain appears to provide the driving force for the molecular movement around the gel fiber.

Electrophoretic size separations in liquified agarose of polystyrene particles and circular DNA

“…Band spacing in agarose gels has been investigated both theoretically [8-111 and experimentally [13]; however, factors affecting the band width have not been investigated quantitatively. In this study, we adopted the rep-…”

Effect of the matrix on DNA electrophoretic mobility

“…Fluorescence videomicroscopy studies have suggested that large DNA molecules become hooked around obstacles in the agarose gel matrix during electrophoresis, leading to U-shaped conformations with the arms of the “U” pointing in the direction of the electric field [59-64]. Eventually, one of the arms of the “U” slides off the obstacle and the DNA collapses into a globular shape, before beginning the stretching and hooking process again [60-63].…”

“…Eventually, one of the arms of the “U” slides off the obstacle and the DNA collapses into a globular shape, before beginning the stretching and hooking process again [60-63]. Since the duration of interaction between DNA and the gel fibers is proportional to DNA length [64], DNA mobilities appear to be determined in part by interactions between the DNA molecules and the agarose gel matrix. Agarose fibers are known to bind anions [65], some of which appear to be localized near the junction zones [66].…”

Stretching and overstretching of DNA in pulsed field gel electrophoresis. I. A quantitative study from the steady state birefringence decay

“…It can now be directly observed by fluorescence videomicroscopy. 12 We show in this paper that the measurements of the overall average of the orientation over many chains, as deduced from dichroism or birefringence, allows to very quantitatively characterize the relative importance of "stretching" and "overstretching" and of their respective dynamics. This characterization relies on the design of an instrument with very high signalto-noise ratio and use of convenient shape to which the birefringence decay can be fitted as described in Materials and Methods.…”