METHOD FOR THE SIMULTANEOUS DETECTION OF MUTATIONS OCCURRING IN RELATED GENOMES, EXPLOITING MOBILITY VARIATIONS IN 2-D ELECTROPHORESIS OF DNA DUPLEXES UNDERGOING DIFFERENTIAL HELIX-COIL TRANSITION IN A DENATURING GRADIENT

摘要

A method for the simultaneous mutation detection exploiting the variable electrophoretic mobility of related DNA samples, caused by the differential onset of helix-coil transition in denaturing gradients, comprises the following subsequent steps: a) preparation of DNA samples; b) fragmentation of the samples through a first restriction; c) terminal labeling of the fragments of each genome with a different fluorochrome; d) mixing the samples; e) carrying out a simultaneous second restriction, converting each long duplex into a set of shorter fragments with two of them labeled at only one of their ends e) carrying out a 2D separation of the mixed digestion products in order to obtain: a first dimension consisting of a standard 20 polyacrylamide gel; and a second dimension against a denaturing gradient; f) spotting, picking of singly labeled spots and sequencing of their DNA.