Open AccessTn5Map, a transposon for the rapid mapping of restriction sites in plasmids
Author(s) -
Hiller Bernhard,
Frey Bruno,
Schumann Wolfgang
Publication year - 1994
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1994.tb06630.x
Subject(s) - plasmid , restriction enzyme , transposable element , restriction map , biology , agarose , cleavage (geology) , hindiii , restriction digest , transposition (logic) , endonuclease , genetics , restriction site , microbiology and biotechnology , restriction fragment , bacterial conjugation , dna , gene , genome , computer science , paleontology , fracture (geology) , artificial intelligence
Abstract A transposon was constructed allowing the rapid restriction mapping of plasmids. This transporon, Tn5Map, contains a cleavage site for the I‐ Sce I endonuclease which recognizes an 18‐mer. After iivo transposition of Tn5Map into the plasmid of interest, the plasmid is isolated and linearized with I‐ Sce I. Splinkers labelled with digoxygenin and complementary to the left and right end of the linearized molecule are added and ligated. After partial digestion of the splinkered molecules with the restriction enzyme of interest, separation of the cleavage products in an agarose gel, and Southern transfer, the labelled fragments are visualized by the addition of the chemiluminescent substrate AMPPD and alkaline phosphatase. The restriction map can be directly read from the bottom to the top of the gel.