SIMPLE AND EFFICIENT GENERATION IN-VITRO OF NESTED DELETIONS AND INVERSIONS - TN5 INTRAMOLECULAR TRANSPOSITION

We have exploited the intramolecular transposition preference of the T n5 in vitro transposition system to test its effectiveness as a tool f or generation of nested families of deletions and inversions. A synthe tic transposon was constructed containing an ori, an ampicillin resist ance (Amp(r)) gene, a multi-cloning site (MCS) and two hyperactive end sequences. The donor DNA that adjoins the transposon contains a kanam ycin resistance (Kan(r)) gene. Any Amp(r) replicating plasmid that has undergone a transposition event (Kan(s)) will be targeted primarily t o any insert in the MCS, Two different size targets were tested in the in vitro system. Synthetic transposon plasmids containing either targ et were incubated in the presence of purified transposase (Tnp) protei n and transformed. Transposition frequencies (Amp(r)/Kan(s)) for both targets were found to be 30-50%, of which >95% occur within the target sequence, in an apparently random manner, By a conservative estimate 10(5) or more deletions/inversions within a given segment of DNA can b e expected from a single one-step 20 mu l transposition reaction. Thes e nested deletions can be used for structure-function analysis of prot eins and for sequence analysis. The inversions provide nested sequenci ng templates of the opposite strand from the deletions.