Importance of different variables for enhancing in situ detection of PCR-amplified DNA.

This study determined the effects of several variables on the in situ signal after PCR amplification in fixed cells. A signal was evident in all human peripheral blood monocytes fixed in buffered formalin using primers for the human proto-oncogene bcl-2 with in situ PCR only after prolonged fixation and protease digestion. A much lower detection rate was noted after ethanol or acetone fixation due to loss of amplified product out of the nucleus into amplifying solution. This observation demonstrates the importance of cross-linking fixatives for retention of amplified DNA at the site of origin. The increased amount of target-specific DNA synthesis evident with the manual hot start modification to in situ PCR was also seen with chemical hot start mediated by the Escherichia coli single-stranded DNA-binding protein. The manual hot start method strongly suppressed in situ unwanted DNA synthesis dictated by nonsense primers; residual nonspecific synthesis was influenced by annealing temperatures and post-fixation protease digestion conditions.