Patent ID: 8735064
Filing Date: 2014-05-27
Classification: C12N,C12Q

Abstract:
1. 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: (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: (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.