Gel Electrophoresis of Nucleic Acids - Factors Affecting Migration of Nucleic Acids

Factors Affecting Migration of Nucleic Acids

The most important factor is the length of the nucleic acid molecule, smaller molecules travel faster, except in field inversion, where it is possible to have "band inversion" - large molecules travel faster than small molecules. But conformation of the nucleic acid molecule, such as % single strand, supercoiling, etc., is also a factor. When analyzing molecules by size, it is most convenient to analyze only linear molecules to avoid this problem, e.g. DNA fragments from a restriction digest, linear DNA PCR products, or RNAs. Agarose gel electrophoresis is widely used to resolve circular DNA with different supercoiling topology, and to resolve fragments that differ due to DNA synthesis (Fangman work).

DNA damage due to increased cross-linking will dose-dependently reduce electrophoretic DNA migration.

Increasing the agarose or polyacrylamide concentration of a gel reduces the migration speed and enables separation of smaller nucleic acid molecules. The higher the voltage, the faster the DNA moves. But voltage is limited by the fact that it heats the gel and ultimately causes it to melt. High voltages also decrease the resolution (above about 5 to 8 V/cm).

Conformations of a DNA plasmid that has not been cut with a restriction enzyme will move with different speeds (slowest to fastest: nicked or open circular, linear, or supercoiled plasmid).