Patent ID: 8735064

Claim:
A method for producing at least one hairpin DNA molecule comprising a loop and a substantially double-stranded stem, said double-stranded stem comprising a double-stranded polynucleotide derived from an mRNA molecule that is a target for at least one small interfering RNA (siRNA) molecule or a candidate siRNA molecule, the method comprising: (a) providing: i) a double-stranded cDNA corresponding to the mRNA or a portion of the mRNA; and ii) at least one hairpin-adapter oligonucleotide comprising a loop and a substantially double-stranded stem, the stem comprising a first endonuclease recognition site that is recognized by a first endonuclease characterized by a cleavage site at least 22 base pairs distant from the recognition site; (b) fragmenting said cDNA to produce at least one cDNA fragment at least 22 base pairs in length, said fragment comprising first and second termini; (c) enzymatically ligating: (i) the first hairpin-adapter oligonucleotide to the first or second terminus of the cDNA fragment, or (ii) the hairpin adapter oligonucleotides to both the first and second termini of the cDNA fragment, to form an intermediate DNA molecule; (d) enzymatically digesting said intermediate DNA molecule with said first endonuclease, thereby producing at least one hairpin DNA molecule comprising a stem comprising at least 22 nucleotide base pairs that correspond to a small interfering RNA (siRNA) that is specific for a target mRNA; (e) treating the hairpin DNA molecule with a DNA phosphatase, thereby producing a dephosphorylated hairpin DNA molecule lacking a 5′ terminal phosphate; (f) providing a linearized vector adaptor and enzymatically ligating the vector adaptor and the dephosphorylated hairpin DNA molecule to produce a fusion nucleic acid comprising a stem-loop and a single-strand nick between the vector adaptor and the 5′ terminus of the hairpin DNA molecule; (g) providing a strand-displacing DNA polymerase and initiating single-strand polymerization of the hairpin DNA moiety by the DNA polymerase at the site of the single strand nick, thereby removing said loop secondary structure; and (h) enzymatically self-ligating the fusion nucleic acid, thereby producing a circularized vector comprising an shRNA template.