Temporal TGGE

Abstract

Recently a modified TGGE system (13, 14, 8) has been developed in which the spatial parallel temperature gradient (from top to bottom) has been replaced with a temporal temperature gradient. The apparatus used for this application (Hoefer Scientific Instruments, San Francisco, USA) is a normal slab gel instrument which can be thermostated accurately at a unique temperature over the whole gel, by placing the frame with the gel in a heated buffer tank. The composition of the gel is identical to that described for TGGE. The glass plates are fixed in an acrylic frame, and after pouring the gel the frame is placed in a heated buffer tank. This gel system has been applied most successfully in combination with psoralen-modified PCR products. In the first stage of electrophoresis, crosslinked fragments may be separated from non-crosslinked fragments. During a one-hour run at a temperature above strand separation of the non-crosslinked nucleic acid, the denatured psoralen-crosslinked molecules migrate much more slowly than the corresponding non-crosslinked single-stranded molecules. In the second stage of electrophoresis, the buffer in the tank is cooled down very slowly (c. 2 h) to a temperature below that required for the reversible transition of the heteroduplexes, and the homo- and heteroduplexes renature and increase their mobility. The resulting band pattern is similar to the one obtained with parallel TGGE. This technique allows the simultaneous analysis of mutations in different PCR products, even if these are of different sequence, length and stability. The electrophoresis is started at a temperature above the T_m of strand separation of the most stable crosslinked DNA fragment, and slowly cooled down below the Tm of the least stable DNA fragment.