Patent ID: 7524653

Claim:
A method for preparing a small interfering RNA (siRNA) library, the method comprising: (1) generating a population of oligoDNAs, each of which comprises a random or semirandom sequence flanked by a 3′ restriction site and a 5′ restriction site, wherein the 3′ restriction site is different than the 5′ restriction site, (2) cloning the oligoDNAs into plasmids having two recombinase sites oriented toward each other so as to orient the random sequence flanked by the 3′ restriction site and the 5′ restriction site between the dual recombinase sites; (3) replicating the plasmids within a population of host cells that produce the appropriate recombinase enzyme for the particular recombinase recognition sites present in the plasmids; (4) exposing the plasmids to the recombinase enzyme within the host cells; (5) isolating plasmid DNA from the population of host cells and digesting the isolated plasmids with a restriction enzyme specific for either the 3′ restriction site or the 5' restriction site; (6) autoligating fragments digested in step (5) which contain one or more dual cassettes wherein a dual cassette comprises inverted repeats of the oligoDNA, which encodes random or semirandom siRNA, wherein the autoligation (step (6)) results in the formation of a tandem inverted repeat of an oligoDNA wherein the repeat sequences are separated by a site for the restriction enzyme used in the digestion of step (5) and flanked by sites for the restriction enzyme used in the digestion of step (7), (7) digesting the autoligated fragments with one or more restriction enzymes specific for the restriction sites flanking the inverted repeats to release the dual cassettes as a population of dual cassettes, and cloning the population of dual cassettes into an RNA expression vector system, which includes an RNA expression vector having an RNA polymerase promoter and an RNA polymerase termination sequence, such that the dual cassettes are inserted between the RNA promoter and the termination sequence; (8) pooling the vectors to form an siRNA-encoding library.