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Nucleic Acids Research, 1988, Vol. 16, No. 3 925-939
© 1988

Articles

Characterization of the ‘unusual’ mobility of large circular DNAs in pulsed field-gradient electroplioresis

Stephen M. Beverley

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School Boston, MA 02115, USA

Received October 21, 1987. Accepted December 14, 1987.

Large circular amplified DNAs (30 and 85 kb) present in methotrexate-resistant Leishinpnja major appear to migrate anomalously in pulsed field-gradient electrophoresis (PFGE), exhibiting pulse time-dependent mobility and migrating along a different apparent path relative to the large linear chromosomal DNAs. Quantitative studies indicate that the relative pulse-time dependence is actually conferred by the mobility properties of the large linear DNAs. One contributing factor to the difference in migration path is variability in the intrinsic voltage-dependence of mobility of supercoiled and linear DNAs, in combination with the asymmetrical/inhomogeneous voltage gradients. Certain linear chromo somes exhibit a previously undescribed pulse-time dependence in the voltage-dependence of mobility. When enzymatically relaxed or physically nicked the large circular DNAS fail to leave the well using any pulse time, a property also observed in conventional electrophoresis. These findings are relevant to PFGE theory, and its application to the study of circular DNA amplification in Leishmania and other species.


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