Polyacrylamide gels are used to analyze and prepare fragments of DNA less than 1 kb in length[107]. They may be cast in a variety of polyacrylamide concentrations, ranging from 3.5% to 20%, depending on the sizes of the fragments of interest (see table tab:page).
In the presence of free radicals, which are usually supplied by ammonium persulfate and stabilized by TEMED (N,N,N',N'-tetramethylenediamine), a chain reaction is initiated in which monomers of acrylamide are polymerized into long chains. When the bifunctional agent N,N'-methylenebisacrylamide is included in the polymerization reaction, the chains become cross-linked to form a gel, whose porosity is determined by the length of the chains and the degree of cross-linking.
The length of the chains is determined by the concentration of acrylamide in the polymerization reaction (between 3.5% and 20%): one molecule of cross-linker is included for every 29 monomers of acrylamide. Polyacrylamide gels are most commonly poured between two glass plates that are held apart by spacers. In this arrangement, most of the acrylamide solution is shielded from the exposure to the air, so that inhibition of polymerization by oxygen is confined to a narrow layer at the top of the gel. Sequencing gels are invariably run in the vertical position, whereas cleangels are run horizontally.
Polyacrylamide gels can range in length from 10 cm to 100 cm, depending on the separation required. However, they have three major advantages over agarose gels: Their resolving power is so great that they can separate molecules of DNA whose lengths differ by as little as 0.2% (i.e., 1 bp in 500 bp). They can further accommodate much larger quantities of DNA than agarose gels, and DNA recovered from polyacrylamide gels is extremely pure.
Sequencing gels (see section seqgel) are polymerized in the presence of an agent (urea ) that suppresses base pairing of nucleic acids. Denatured DNA migrates through gels at a rate that is almost completely independent of its base composition and sequence.