Abstract: Template specific inhibition during PCR (TSI-PCR) allows specific inhibition of particular templates without disrupting the amplification of other templates that share amplification primer binding sites. TSI-PCR is achieved by ligation of stop oligos comprising a 3? modification that prevents extension by DNA polymerases. Stop oligos hybridize to a region of the targeted amplicon downstream of the amplification primer. When the stop oligos are perfectly complementary to the target template, they are ligated onto the extending strand during the extension phase of the amplification cycle. This effectively truncates the extending strand at the site where the stop oligo binds and blocks further extension. The truncated products are not full-length and cannot be extended, and therefore do not serve as additional templates during sunsequent rounds of amplification. This results in substantial inhibition over multiple amplification cycles, but only for templates that match the stop oligos.