A number of factors can affect the migration of nucleic acids: the dimension of the gel pores, the voltage used, the ionic strength of the buffer, and the concentration intercalating dye such as ethidium bromide if used during electrophoresis.[1]
Size of DNA
The gel sieves the DNA by the size of the DNA molecule whereby smaller molecules travel faster. Double-stranded DNA moves at a rate that is inversely proportional to the log10 of the number of base pairs. This relationship however breaks down with very large DNA fragments and it is not possible to separate them using standard agarose gel electrophoresis. The limit of resolution depends on gel composition and field stength.[2] and the mobility of larger circular DNA may be more strongly affected than linear DNA by the pore size of the gel.[3] Separation of very large DNA fragments requires pulse field gel electrophoresis (PFGE). In field inversion gel electrophoresis (FIGE, a kind of PFGE), it is possible to have "band inversion" - where large molecules may move faster than small molecules.
Gels of plasmid preparations usually show a major band of supercoiled DNA with other fainter bands in the same lane. Note that by convention DNA gel is displayed with smaller DNA fragments near the bottom of the gel. This is because historically DNA were run vertically and the smaller DNA fragments move downwards faster.
Conformation of DNA
The conformation of the DNA molecule can significantly affect the movement of the DNA, for example, supercoiled DNA usually moves faster than relaxed DNA because it is tightly coiled and hence more compact. In a normal plasmid DNA preparation, multiple forms of DNA may be present,[4] and gel from the electrophoresis of the plasmids would normally show a main band which would be the negatively supercoiled form, while other forms of DNA may appear as minor fainter bands. These minor bands may be nicked DNA (open circular form) and the relaxed closed circular form which normally run slower than supercoiled DNA, and the single-stranded form (which can sometimes appear depending on the preparation methods) may move ahead of the supercoiled DNA. The rate at which the various forms move however can change using different electrophoresis conditions, for example linear DNA may run faster or slower than supercoiled DNA depending on conditions,[5] and the mobility of larger circular DNA may be more strongly affected than linear DNA by the pore size of the gel.[6] Unless supercoiledDNA markers are used, the size of a circular DNA like plasmid therefore may be more accurately gauged after it has been linearized by restriction digest.
DNA damage due to increased cross-linking will also reduce electrophoretic DNA migration in a dose-dependent way.[7][8]
Concentration of ethidium bromide
Circular DNA are more strongly affected by ethidium bromide concentration than linear DNA if ethidium bromide is present in the gel during electrophoresis. All naturally occurring DNA circles are underwound, but ethidium bromide which intercalates into circular DNA can change the charge, length, as well as the superhelicity of the DNA molecule, therefore its presence during electrophoresis can affect its movement in gel. Increasing ethidium bromide intercalated into the DNA can change it from a negatively-supercoiled molecule into a fully relaxed form, then to positively-coiled superhelix at maximum intercalation.[9] Agarose gel electrophoresis can be used to resolve circular DNA with different supercoiling topology.
Gel concentration
The concentration of the gel determines the pore size of the gel which affect the migration of DNA. The resolution of the DNA changes with the percentage concentration of the gel. Increasing the agarose concentration of a gel reduces the migration speed and improves separation of smaller DNA molecules, while lowering gel concentration permits large DNA molecules to be separated. For a standard agarose gel electrophoresis, a 0.7% gives good separation or resolution of large 5–10kb DNA fragments, while 2% gel gives good resolution for small 0.2–1kb fragments. Up to 3% can be used for separating very tiny fragments but a vertical polyacrylamide gel would be more appropriate for resolving small fragments. High concentrations gel however requires longer run times (sometimes days) and high percentage gels are often brittle and may not set evenly. High percentage agarose gels should be run with PFGE or FIGE. Low percentage gels (0.1 - 0.2%) are fragile and may break. 1% gels are common for many applications.[10]
Applied field
At low voltages, the rate of migration of the DNA is proportional to the voltage applied, i.e. the higher the voltage, the faster the DNA moves. However, in increasing electric field strength, the mobility of high-molecular-weight DNA fragments increases differentially, and the effective range of separation decreases and resolution therefore is lower at high voltage. For optimal resolution of DNA > 2kb in size in standard gel electrophoresis, 5 to 8 V/cm is recommended.[5] Voltage is also limited by the fact that it heats the gel and may cause the gel to melt if a gel is run at high voltage for prolonged period, particularly for low-melting point agarose gel.
The mobility of DNA however may change in an unsteady field. In a field that is periodically reversed, the mobility of DNA of a particular size may drop significantly at a particular cycling frequency.[11] This phenomenon can result in band inversion whereby larger DNA fragments move faster than smaller ones in PFGE.