RAPID DISCRIMINATION OF SEQUENCES FLANKING AND WITHIN T-DNA INSERTIONS IN THE ARABIDOPSIS GENOME

An improvement to previous methods for recovering Arabidopsis thaliana genomic DNA flanking T-DNA insertions is presented that allows for th e avoidance of some of the cloning difficulties caused by the concatam eric nature of T-DNA inserts. The principle of the procedure is to cat egorize by size restriction fragments of mutant DNA, produced in separ ate digestions with Ndel and Bst1107I. Given that the sites for these two enzymes are contiguous within the pGV3850:1003 T-DNA construct, th e restriction fragments obtained fall into two categories: those showi ng identical size in both digestions, which correspond to sequences in ternal to T-DNA concatamers; and those of different sizes, that contai n the junctions between plant DNA and the T-DNA insert. Such a criteri on makes it possible to easily distinguish the digestion products corr esponding to internal T-DNA parts, which do not deserve further attent ion, and those which presumably include a segment of the locus of inte rest. Discrimination between restriction fragments of genomic mutant D NA can be made on rescued plasmids, inverse PCR amplification products or bands in a genomic blot.