Mr. Ponce et al., RAPID DISCRIMINATION OF SEQUENCES FLANKING AND WITHIN T-DNA INSERTIONS IN THE ARABIDOPSIS GENOME, Plant journal, 14(4), 1998, pp. 497-501
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.