TN5 IN-VITRO TRANSPOSITION

This communication reports the development of an efficient in vitro tr ansposition system for Tn5. A key component of this system was the use of hyperactive mutant transposase. The inactivity of wild type transp osase is likely to be related to the low frequency of in vivo transpos ition. The in vitro experiments demonstrate the following: the only re quired macromolecules for most of the steps in Tn5 transposition are t he transposase, the specific 19-bp Tn5 end sequences, and target DNA; transposase may not be able to self-dissociate from product DNAs; Tn5 transposes by a conservative ''cut and paste'' mechanism; and Tn5 rele ase from the donor backbone involves precise cleavage of both 3' and 5 ' strands at the ends of the specific end sequences.