You must have known that agarose gel electrophoresis is generally adequate for resolving nucleic acid fragments in the size range of 100 nucleotides to around 10-15 kb. But, for nucleic acid which its fragments below those range, it will be difficult to separate and hard to visualize because of diffusion within the gel matrix. These problems are solved by native polyacrylamide gel electrophoresis (PAGE). Using native PAGE, fragments as small as 10 bp and up to 1 kb can be separated with a resolution of as little as 1 bp.
Polyacrylamide Gel Electrophoresis has a number of advantages, which are:
- PAGE has a high loading capacity, up to 10 micrograms of DNA can be loaded into a single well (1 cm x 1 mm) without significant loss of resolution.
- Polyacrylamide contains few inhibitors of enzymatic reactions.
- PAGE is an ideal gel system from which to isolate DNA fragments for subcloning and other molecular biological techniques.
As any other methods, PAGE also has disadvantages:
- The mobility of the fragments can be affected by base composition making accurate sizing of bands a problem.
- Polyacrylamide quenches fluorescence, making bands containing less than 25 ng difficult to visualize with ethidium bromide staining.
Note: As an alternative method for visualizing DNA fragments instead of using Ethidium Bromide staining is carried out by wrapping the gel in a UV-transparent plastic film, such as Saran Wrap, and then placing it onto a thin-layer chromatography plate that contains a UV fluorescent indicator. Long-wave UV light is shone through the gel onto the chromatography plate, causing it to glow. Regions of high DNA concentration leave a “shadow” on the plate as the transmitted UV is absorbed by the DNA. The position of the DNA can be marked on the Saran Wrap with a fiber-tip pen and then cut from the gel.
The methods to separate and to purify DNA fragments will be described later in the next post.