Simple and efficient generation in vitro of nested deletions and inversions: Tn5 intramolecular transposition.
We have exploited the intramolecular transposition preference of the Tn 5 in vitro transposition system to test its effectiveness as a tool for generation of nested families of deletions and inversions. A synthetic transposon was constructed containing an ori, an ampicillin resistance (Ampr) gene, a multi-cloning site (MCS) and two hyperactive end sequences. The donor DNA that adjoins the transposon contains a kanamycin resistance (Kanr) gene. Any Amprreplicating plasmid that has undergone a transposition event (Kans) will be targeted primarily to any insert in the MCS. Two different size targets were tested in the in vitro system. Synthetic transposon plasmids containing either target were incubated in the presence of purified transposase (Tnp) protein and transformed. Transposition frequencies (Ampr/Kans) 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 be expected from a single one-step 20 microl transposition reaction. These nested deletions can be used for structure-function analysis of proteins and for sequence analysis. The inversions provide nested sequencing templates of the opposite strand from the deletions.
York
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D
Department of Biochemistry, 420 Henry Mall, University of Wisconsin-Madison, Madison, WI 53706, USA.
Welch
K
K
Goryshin
I Y
IY
Reznikoff
W S
WS
D007446:Chromosome Inversion; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D007613:Kanamycin Resistance / Q000235:genetics; D016254:Mutagenesis, Insertional / Q000379:methods*; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D018741:Replication Origin; D015183:Restriction Mapping; D017384:Sequence Deletion
D004251:DNA Transposable Elements