DNA sequencing with direct blotting electrophoresis and colorimetric detection

Abstract

We describe optimized procedures for colorimetrically-detected DNA sequencing with direct blotting electrophoresis. One-step protocols for Sequenase and Klenow enzyme are given. The clapping technique has been adapted to allow convenient casting of very thin gels with an optimal lower gel (transfer) surface. This gives very sharp band patterns, enabling more than 350 bases from a single loading to be read with confidence. The crucial points for direct blotting electrophoresis are discussed. Background problems resulting from unspecific binding of streptavidin to the nylon membranes have been eliminated by the use of high concentrations of SDS in the incubation buffer; and using a single large glass tube for all incubation and washing steps is a very convenient and effective development protocol. Automation of the colorimetric development process is described.