Direct Blotting Electrophoresis

Abstract

The analysis of membrane-immobilized macromolecules such as DNA/RNA and proteins presents a common analytical method with widespread applications. The macromolecules are either spotted directly onto a membrane or, if they are a complex mixture, they are separated by electrophoresis prior to the transfer. Over the last 15 years a variety of procedures and equipment has been developed to achieve such transfer or blotting of gel-separated macromolecules onto an immobilizing matrix. The first transfer method was described by Southern in 1975 and was based on capillary action (1). Since then, procedures have been reported based on electro, vacuum, and pressure blotting, among them direct blotting electrophoresis (DBE) (2). DBE requires special equipment but it allows to combine electrophoresis and electroblotting into a single procedure. The principle of DBE is shown schematically in Fig. 1.. The same electric field that separates applied macromolecules in one dimension is utilized for electro-blotting them onto an immobilizing matrix that is moved across the bottom of the gel by a conveyor belt. DBE has been used for Southern blots, Western blots, DNA sequencing, and other applications (2–11). Since this volume is dedicated to DNA sequencing, the following chapter will be limited to the use of DBE for this particular application.