Patent ID: 8293974

Claim:
A method of constructing a plant expression construct comprising a plant expressible promoter operably linked to a DNA sequence encoding a mismatched long double-stranded RNA (IdsRNA) duplex, the method including the steps of: (i) amplifying a DNA fragment of a target gene to produce an untreated fragment; (ii) treating a fraction of the amplified DNA fragment of the target gene with a bisulphite mutagen to produce a treated fragment, thereby causing random chemical mutation of cytosine nucleotides to thymine nucleotides, thus creating a proportion of mismatched bases in the treated fragment compared with the untreated fragment; (iii) conducting sense strand-specific PCR on the treated fragment from (ii); (iv) conducting PCR on the untreated fragment from (i); (v) ligating the PCR amplified dsDNA fragments from (iii) and (iv) in a head-to-head orientation such that the two ligated dsDNA fragments are adjacent and in an inverted orientation to each other with respect to their sense strands; and (vi) operably linking a plant-expressible promoter to the DNA sequence encoding a mismatched IdsRNA duplex of part (v).