Polyacrylamide gels are used to analyse and prepare fragments of DNA less than 1 kb in length [119]. 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.
Polyacrylamide gels are formed by co-polymerisation of acrylamide and N,N’-methylenebisacrylamide. The reaction is a vinyl addition polymerisation initiated by a free radical generating system:
Polymerisation is initiated by TEMED (N,N,N’,N’-tetramethylenediamine) and APS (ammonium persulfate). The TEMED acts as an electron carrier to activate the acrylamide monomer, providing an unpaired electron to convert the acrylamide monomer to a free radical.
The activated monomer then reacts with an unactivated monomer to begin the polymer chain reaction. The elongating polymer chains are randomly crosslinked by bisacrylamide, resulting in closed loops and a complex web polymer with a reproducible porosity that depends on the polymerisation conditions and monomer concentration.
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 4.2.7) are polymerised 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.
For Direct Sequencing (see 4.2.8) 4.25% 19:1 polyacrylamide gels (6 M urea) and 5% 29:1 polyacrylamide gels (6 M urea) have been used.
© 2001 Alexander Binder