A PCR-based strategy for cloning short hairpin sequences:“PCR SHAGging”.

Our overall approach is to use an RNA polymerase III promoter to drive expression of encoded short hairpin RNA (shRNA). For this purpose we use the human U6 snRNA promoter, maintaining the transcript initiating “G” nucleotide of the U6snRNA transcript. There by, hairpin sequences will start with a “G”. Termination is mediated by a run of Ts at the end of the hairpin. We have found hairpins of 27 to 29nt in length to be as effective, if not more so, than hairpins containing 19nt and 21nt stems. Thereby, our hairpins currently contain 29nt of stem. An additional design feature is the inclusion of a few G-U pairings in the hairpin stem (which are permitted in dsRNA alpha helices) to stabilize hairpins during propagation in bacteria. We are continuing to test length and structural features of our hairpins and will up date our web site accordingly.