Nucleic Acids Research, 2002, Vol. 30, No. 21 e119
© 2002 Oxford University Press
Production of combinatorial libraries of fused genes by sequential transposition reactions
Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA
*To whom correspondence should be addressed. Tel: +1 608 262 3608; Fax: +1 608 265 2603; Email: reznikoff{at}biochem.wisc.edu
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
The use of in vivo and in vitro transposition reactions to perform non-combinatorial manipulation of DNAs in molecular biology is widespread. In this work we describe a technique that utilizes two sequential, directed transposition reactions in order to carry out combinatorial DNA manipulations. The methodology relies on the use of two different mutant Tn5 transposase proteins that have different transposon end recognition specificities. We demonstrate that the technique can be used to create large libraries of random fusions between two genes. These transpositional fusions are defined by insertion of a 32 bp linker sequence. We applied the technique to a model system, chloramphenicol acetyl transferase, to create functional fusions from N- and C-terminally truncated, non-functional genes. Comparative structural analysis suggests that both sides of the linker are inserted into disordered regions in functional proteins.