Detection of genomic DNA with a high sensitivity in tissue sections using a two step cycling in situ PCR procedure

In situ hybridization has been used for the detection of viral and bacterial mRNA and DNA which has infected tissue sections, but the sensitivity of this method is very limited. In this paper, a new, sensitive and simple non-isotopic in situ hybridization method in combination with a polymerase chain reaction (PCR) technique is described, in which the genomic or viral DNA can be amplified directly on formalin-fixed and paraffin-embedded tissue sections. As low as two copies of target DNA per cell can be detected by this in situ hybridization after in situ PCR. The essential modification concerns a two step cycling PCR procedure that was applied using single primer pairs. The first step, annealing, was achieved at a low temperature for a relatively long time. The second step was performed at a high temperature for a short period of time. This procedure greatly decreased the non-specific amplification of DNA and increased the sensitivity of detection.