Described herein is a method of
cloning synthetic oligos (including in situ synthesized oligos) into an (one or more)
expression vector for
library (e.g., shRNA
library) production. The oligos are synthesized with one portion of the first stem of the hairpin, followed by a first loop sequence, the complete second stem, a second loop sequence, and finished with the remaining portion of the first stem of the hairpin. The two portions of the first stem anneal to the second stem, juxtaposing the 5′ end close to the 3′ end of the oligo. The methods described herein selected for hairpins with perfectly base-paired stems. After annealing, a ligase is added to the annealed oligos and the base-paired hairpins are preferentially annealed, and ligated, creating closed circular oligos. The now circularized hairpins served as templates for rolling circle amplification using a
polymerase with high
processivity. One or more primers complementary to the two strands of the amplified
double stranded circular hairpins initiate the rolling circle amplification in the presence of a
polymerase. Using primers (e.g., a sense and antisense primer), the rolling circle amplification yields
double stranded hairpin sequences. These can be digested (e.g., using restriction enzymes) to produce a double-stranded hairpin fragment encoding a single hairpin. The fragment can be cloned into an appropriately digested vector for a variety of uses including expression.