Electrophoretic Fragment Separation

Electrophoretic Fragment Separation

The phosphate backbones of DNA and RNA molecules give them a uniform charge per unit length. Therefore, upon electrophoresis through polyacrylamide or agarose gels, molecules will migrate at rates largely independent of their sequences. The frictional or retarding forces the gels exert on the migrating molecules increase sharply with the length of the DNA or RNA so that the larger the molecule, the slower it migrates through a gel. This is the basis of the exceptionally valuable technique of electrophoresis. In general, two molecules whose sizes differ by 1% can be separated. Polyacrylamide gels are typically used for molecules from five to perhaps 5,000 base pairs, and agarose gels are used for molecules 1,000 base pairs and larger.

Following electrophoresis, the locations of specific DNA fragments can be located by staining or by autoradiography. Ethidium bromide is a most useful stain for this purpose. The molecule is nonpolar an readily intercalates between bases of DNA. In the nonpolar environment between the bases, its fluorescence is increased about 50 times. There-fore a gel can be soaked in a dilute solution of ethidium bromide and illumination with an ultraviolet lamp reveals the location of DNA as bands glowing cherry-red. As little as 5 ng of DNA in a band can be detected by this method. For detection of smaller quantities of DNA, the DNA can be radioactively labeled before electrophoresis. A simple enzymatic method of doing this is to use the enzyme polynucleotide kinase to transfer a phosphate group from ATP to the 5’-OH of a DNA molecule. After electrophoresis, a radioactive DNA band is located by exposing a photographic film to the gel and developing. The radioactive decay of the ³²P sensitizes the silver halide crystals in the film so that upon development, black particles of silver remain to reveal positions of radioactive DNA or RNA in the gel.

Above about 50,000 base pairs long, all DNA migrates in gels at about the same rate. This results from the DNA assuming a conformation in which its charge to frictional force ratio is independent of its length as it snakes through the gel in a reptilian fashion. It was empirically found, however, that brief periodic changes in the direction of the electric field or polarity reverses often will separate still larger DNA molecules. This techniques is called pulsed field electrophoresis. Overall, the major motion of the DNA is in one direction, but it is punctuated by reversals or changes in direction from once per second to once per minute. The change in migration direction destroys the structure of the species whose migration rates are independent of size, and for a short while, the long DNA molecules migrate at rates related to their sizes. Additional size separation is achieved in these electrophoretic techniques because the larger the molecule, the longer it takes to achieve the steady-state snaking state. By these means, molecules as large as 1,000,000 base pair chromosomes can be separated by size.