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Rapid discrimination of sequences flanking and within T‐DNA insertions in the Arabidopsis genome
Author(s) -
Ponce María Rosa,
Quesada Víctor,
Micol José Luis
Publication year - 1998
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.1998.00146.x
Subject(s) - arabidopsis , genome , genetics , dna , biology , computational biology , flanking maneuver , dna sequencing , gene , mutant , history , archaeology
Summary An improvement to previous methods for recovering Arabidopsis thaliana genomic DNA flanking T‐DNA insertions is presented that allows for the avoidance of some of the cloning difficulties caused by the concatameric nature of T‐DNA inserts. The principle of the procedure is to categorize by size restriction fragments of mutant DNA, produced in separate digestions with Nde I and Bst 1107I. Given that the sites for these two enzymes are contiguous within the pGV3850:1003 T‐DNA construct, the restriction fragments obtained fall into two categories: those showing identical size in both digestions, which correspond to sequences internal to T‐DNA concatamers; and those of different sizes, that contain the junctions between plant DNA and the T‐DNA insert. Such a criterion makes it possible to easily distinguish the digestion products corresponding to internal T‐DNA parts, which do not deserve further attention, and those which presumably include a segment of the locus of interest. Discrimination between restriction fragments of genomic mutant DNA can be made on rescued plasmids, inverse PCR amplification products or bands in a genomic blot.