Information on DNA conformation derived from transverse pore gradient gel electrophoresis in conjunction with an advanced data analysis applied to capillary electrophoresis in polymer media.
Abnormally slow migration of DNA is conventionally viewed as being due to an abnormal conformation relative to "linear" standards. The evidence for this rests on a few instances where nonlinear DNA structures have been established by independent methods and yield low mobilities relative to standards. Transverse pore gradient gel electrophoresis of authentically bent kinetoplast DNA and of an upstream activator sequence (UAS) of an E. coli operon promoter shows in addition that curves of migration distance vs. gel concentration ("Ferguson curves") of such abnormally conformed DNA differ from those of "linear" standards. Since Ferguson curves are interpretable with regard to molecular size in concordance with a mathematical model (Ogston model), transverse pore gradient gel electrophoresis provides a simple means of correlating abnormally slow migration of DNA with molecular size. In addition, transverse pore gradient gel electrophoresis is able to distinguish between DNA banding which exhibits a steeper dependence on gel concentration than "linear" standards from one which shows the same dependence. The former appears characteristic of circularly bent DNA and gives rise to a substantial retardation, the latter of bending across a knot or kink in the DNA chain associated with a relatively minor retardation relative to standards. Circularly bent restriction fragments formed from kinetoplast DNA retain the characteristic intersecting Ferguson curves on the transverse pore gradient gel. Another authentically "abnormal" DNA structure recognizable on transverse pore gradient gels is supercoiled DNA derived from the reaction of topoisomerase with a plasmid. Different lengths of supercoiled sequences give rise to parallel Ferguson curves clearly intersecting with those of linear standards.(ABSTRACT TRUNCATED AT 250 WORDS)
Wheeler
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Laboratory of Theoretical and Physical Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-0001.
Tietz
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Chrambach
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D000818:Animals; D004247:DNA / Q000737:chemistry* / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D016054:DNA, Protozoan / Q000737:chemistry / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D009690:Nucleic Acid Conformation; D012984:Software
D004269:DNA, Bacterial; D016054:DNA, Protozoan; D004247:DNA