In situ amplification using universal energy transfer-labeled primers

We developed an amplification detection system in which a universal energy transfer-labeled primer (UniPrimer) is used in combination with any target- specific primer pair. The target specific primers each have a 5' tail seque nce, which is homologous to the 3' end of the UniPrimer which, in turn, has a hairpin structure on the 5' end. The hairpin structure brings the fluoro phore and quencher into close proximity when the primer is free in solution , providing efficient quenching. When the primer is incorporated into the P CR product, the hairpin structure is unfolded and a fluorescent signal can be detected. Using hepatitis C and human papillomavirus as model systems, t his study demonstrates several advantages in the hot-start in situ PCR tech nique with the UniPrimer system, including target specific detection of one DNA copy per cell without a separate in situ hybridization step and detect ion of an RNA target by RT in situ PCR without overnight DNase digestion. T he UniPrimer-based in situ PCR allows rapid and simple detection of any DNA or RNA target without concern for the background from DNA repair invariabl y evident in paraffin-embedded tissue when a labeled nucleotide is used.