Denaturing gel electrophoresis for sequencing

The accuracy of DNA sequence determination depends largely upon resolution of the sequencing products in denaturing polyacrylamide gels. This unit provides a detailed description of the setup, electrophoresis, and processing of such gels. In general, the gels required for DNA sequencing are 40-cm long, of uniform thickness, and contain 4% to 8% acrylamide and 7 M urea. Modifications of this protocol increase the length of readable sequence information which can be obtained from a single gel (i.e., forming the gel with wedge-shaped spacers to create a field gradient, or incorporating a buffer gradient, an electrolyte gradient, or an acrylamide step gradient into the gel). A modification to the Basic Protocol--inclusion of formamide in the sequencing gel--is designed to overcome gel compressions arising from secondary structure in the sequencing products during gel electrophoresis. A discussion of acrylamide concentrations and electrophoresis conditions is included in the Commentary.