Conformational correlatives of DNA band compression and bidirectional migration during field inversion gel electrophoresis, detected by quantitative video epifluorescence microscopy.
Individual DNA molecules in the Mb size range were monitored by epifluorescence video microscopy during field inversion gel electrophoresis (FIGE). DNA migrating in an agarose gel gives rise to characteristic V-conformational elements and when doing so exhibits a reduced mobility. When the V-conformational elements per DNA molecule are few, the degree of retardation appears proportional to the number of V's, and since larger DNA species exhibit more V's, to DNA size. For a particular pulse frequency, the proportionality breaks down progressively as the number of V-conformational elements per DNA molecule increases. The loss of proportionality between DNA length and migration rate is being correlated with the macroscopically observed loss of electrophoretic size discrimination known as band compression. For a particular pulsing frequency and size class of DNA, the loss of size discrimination is thought to be due to the different orientations of migration, caused by the asymmetric distribution of V-conformational elements when the number of these elements is moderate. Small and very large DNA by contrast migrate with the direction of the biased field. These events, analyzed by microscopic measurement, are consistent with the known macroscopically observed double-valued mobilities in FIGE.
Rampino
N J
NJ
Section on Macromolecular Analysis, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Chrambach
A
A
D004247:DNA / Q000737:chemistry*; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods; D008856:Microscopy, Fluorescence; D009690:Nucleic Acid Conformation; D010784:Photomicrography; D014743:Videotape Recording
D004247:DNA