Electrophoresis is an extremely useful procedure when applied to analysis of nucleic acids as it can resolve molecules of different sizes with relative ease and accuracy. Large molecules migrate more slowly than small molecules in agarose gels. However, the fact that nucleic acids of the same length may exist in a variety of conformations can often complicate the interpretation of electrophoretic separations. For instance, when a single species of a bacterial plasmid is isolated from cells, the individual plasmids may exist in three forms (depending on the genotype of their host and conditions of isolation): superhelical/supercoiled (form I), nicked/open circle (form II), and linear (form III). Form I is compact and very tightly coiled, with both DNA strands continuous. Form II exists as a loose circle because one of the two DNA strands has been broken, thus releasing the supercoil. All three have the same mass, but each will migrate at a different rate through a gel. Based on your understanding of gel composition and DNA migration, predict the relative rates of migration of the three DNA structures mentioned above.

Key Concepts

Electrophoresis

Electrophoresis is a laboratory technique used to separate charged molecules, such as nucleic acids, based on their size and charge. In this process, an electric field is applied to a gel matrix, causing molecules to migrate through the gel. Smaller molecules move faster and travel further than larger ones, allowing for the resolution of different sizes. This technique is essential for analyzing DNA fragments and understanding their properties.

DNA Conformations

DNA can exist in various conformations, which affect its physical properties and behavior during electrophoresis. The three primary forms mentioned are supercoiled (form I), nicked/open circular (form II), and linear (form III). Supercoiled DNA is compact and migrates faster due to its tight structure, while nicked and linear forms are less compact, resulting in slower migration rates. Understanding these conformations is crucial for interpreting electrophoretic results.

Gel Composition

The composition of the gel, typically agarose for nucleic acid analysis, influences the separation of DNA molecules during electrophoresis. Agarose concentration affects the pore size of the gel, which in turn impacts the migration rates of different DNA conformations. A higher concentration of agarose creates smaller pores, hindering the movement of larger molecules, while lower concentrations allow for better separation of larger fragments. This knowledge is vital for optimizing electrophoresis conditions.