Patent ID: 7727744

Claim:
A method for generating a host cell transformed with an amplification product encoding a directionally truncated polypeptide, the method comprising: (a) providing: i) a double-stranded target nucleic acid molecule, ii) host cells, and iii) a separate transposon comprising an origin of replication capable of directing replication in the host cells; (b) contacting said target nucleic acid molecule with said transposon under conditions wherein insertion of said transposon into said target nucleic acid molecule occurs, thereby generating a transposon-containing target nucleic acid molecule; (c) amplifying the transposon-containing target nucleic acid molecule by PCR using a first primer that anneals to a target sequence in said target nucleic acid molecule that encodes a polypeptide from which it is desired to generate a directionally truncated polypeptide and a second primer that anneals to said transposon in an opposite polarity to said first primer to generate amplification products that are delimited by said first and second primer, wherein each amplification product comprises at least a portion of the sequence from said transposon, wherein said portion includes the origin of replication, and at least a truncated portion of, and no more than a truncation portion of, said target sequence in said target nucleic acid molecule; (d) ligating said amplification products so that each amplification product is ligated to itself to generate a circular amplification product; (e) transforming said circular amplification products into host cells, wherein each said circular amplification product is replicated in a host cell using said origin of replication of said transposon; and (f) selecting a host cell that contains a replicated circular amplification product that encodes a directionally truncated polypeptide from among the host cells transformed in step (e).