Characterization of the 'unusual' mobility of large circular DNAs in pulsed field-gradient electrophoresis.
Large circular amplified DNAs (30 and 85 kb) present in methotrexate-resistant Leishmania 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 chromosomes 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.
Beverley
S M
SM
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115.
D000818:Animals; D004270:DNA, Circular; D004278:DNA, Superhelical; D004586:Electrophoresis / Q000379:methods*; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D005784:Gene Amplification; D007895:Leishmania tropica / Q000235:genetics
D004270:DNA, Circular; D004278:DNA, Superhelical