Double transposition methods for manipulating nucleic acids

A method for producing a gene fusion library, the method comprising the steps of:

• mixing together, under conditions suitable for in vitro transposition, (1) copies of a transposable polynucleotide that comprises distinct first and second transposase-interacting inverted repeat sequence pairs, members of the first sequence pair flanking members of the second sequence pair, the members of the second sequence pair flanking a first sequence for conferring selectability upon a host cell, (2) copies of a first target nucleic acid molecule that comprises a first polypeptide-encoding gene sequence, a first origin of replication and a second sequence for conferring selectability upon a host cell, and (3) a first transposase that binds to and interacts with the first sequence pair, to produce first transposition products;
• introducing the first transposition products into host cells;
• selecting host cells that comprise first transposition products wherein the first polypeptide-encoding gene sequence is disrupted by the transposable polynucleotide;
• mixing (1) the transposition products from the selected host cells and (2) a second target nucleic acid molecule that comprises a second polypeptide-encoding gene sequence, a second origin of replication and a third sequence for conferring selectability upon a host cell, and (3) a second transposase that binds to and interacts with the second sequence pair, to produce second transposition products;
• introducing the second transposition products into host cells;
• selecting host cells that comprise second transposition products comprising fusions between the first and the second polypeptide-encoding gene sequences.