Related Experiment Videos
Characterization of the 'unusual' mobility of large circular DNAs in pulsed field-gradient electrophoresis
1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115.
Abstract:
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.
Insights
Anomalous DNA migration in pulsed field-gradient electrophoresis (PFGE) was observed for circular amplified DNA in Leishmania major. Linear DNA properties, not circular DNA, primarily influence this complex migration behavior during PFGE.
Area of Science:
- Molecular Biology
- Genetics
- Biophysics
Background:
- Methotrexate-resistant Leishmania major harbors large circular amplified DNAs (30 and 85 kb).
- Pulsed field-gradient electrophoresis (PFGE) is a technique used to separate large DNA molecules.
Purpose of the Study:
- To investigate the anomalous migration of large circular amplified DNAs in Leishmania major using PFGE.
- To elucidate the factors contributing to the differential migration patterns between circular and linear DNAs during PFGE.
Main Methods:
- Pulsed field-gradient electrophoresis (PFGE) was employed to analyze DNA migration.
- Quantitative studies were conducted to assess pulse time-dependent mobility.
- Enzymatic relaxation and physical nicking were used to alter DNA topology.
Main Results:
- Large circular amplified DNAs exhibited anomalous, pulse time-dependent mobility in PFGE, differing from linear chromosomal DNAs.
- Quantitative analysis revealed that linear DNA mobility properties were the primary drivers of the observed pulse-time dependence.
- Variability in voltage-dependence of mobility for supercoiled versus linear DNAs, coupled with gradient asymmetry, influenced migration paths.
- Relaxed or nicked circular DNAs failed to migrate from the electrophoresis well, similar to conventional electrophoresis.
Conclusions:
- The study clarifies PFGE behavior of amplified circular DNA in Leishmania.
- Findings contribute to a deeper understanding of PFGE theory for analyzing circular DNA amplification.
- Results have implications for studying DNA amplification in Leishmania and other species using PFGE.