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Plasmid migration using orthogonal-field-alternation gel electrophoresis
R C Hightower1, D W Metge, D V Santi
1Department of Biochemistry, University of California, San Francisco 94143.
Nucleic Acids Research
|October 26, 1987
Summary
Supercoiled plasmid DNA migration on orthogonal-field-alternation gel electrophoresis (OFAGE) is size-dependent and unaffected by pulse times. Supercoiling significantly impacts migration, enabling distinct separation from linear DNA molecules.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Orthogonal-field-alternation gel electrophoresis (OFAGE) is a technique used for separating large DNA molecules.
- Understanding DNA migration patterns is crucial for molecular biology applications, including DNA analysis and purification.
Purpose of the Study:
- To analyze the migration properties of supercoiled plasmids using OFAGE.
- To investigate the influence of plasmid size, supercoiling, and electrophoresis parameters on migration.
- To explore practical applications of OFAGE for distinguishing circular and linear DNA.
Main Methods:
- Utilized orthogonal-field-alternation gel electrophoresis (OFAGE) to analyze supercoiled plasmids.
- Examined plasmids ranging from 4 to 16 kilobases (kb).
- Compared migration of supercoiled, relaxed, and nicked open circular DNA forms, including pBR322 topoisomers.
Main Results:
- Supercoiled plasmids from 4-16 kb entered the gel and were well resolved by OFAGE.
- Plasmid relative mobilities remained constant across a wide range of pulse times (10-120s) and running times (6-24h).
- The degree of DNA supercoiling significantly altered plasmid migration patterns on OFAGE.
Conclusions:
- OFAGE effectively separates supercoiled plasmids, with migration independent of pulse and running times.
- Supercoiling is a critical factor influencing plasmid mobility in OFAGE.
- The distinct migration behaviors of circular and linear DNA on OFAGE offer practical separation and analysis advantages.