chapter  20
7 Pages

In Situ Amplification of cDNA

The ability to isolate, amplify, and characterize specific DNA and RNA sequences has led to tremendous changes in such diverse fields as basic research, forensic pathology, and the study of infectious disease. In many instances, it is highly desirable to determine what specific cell type contains the DNA or RNA sequence of interest. One example, discussed in this chapter, are the specific targets of infection by the virus HIV-1. In theory, this question could be addressed by in situ hybridization. However, this method requires the presence of at least 10 target sequences in an intact cell in order to produce a detectable signal.1 In many instances only one or a few targets may be present in a given cell. HIV-1 is a classic example, where one copy of the DNA provirus of HIV-1 can integrate into the nucleus of the host cell and remain latent for many years. Solution phase PCR, on the other hand, can detect as few as 1 target per 100,000 cells,2,3 but information about its localization is lost due to the obligatory destruction of cells for DNA extraction. By combining the cell localizing ability of in situ hybridization with the high sensitivity of PCR, one can readily and routinely detect 1 to 10 target sequences in a cell and —either by using its cytologic features or colocalizing probes-determine the exact nature of that cell. Thus, PCR in situ

hybridization (for DNA) and in situ reverse transcriptase (RT) PCR (for RNA) are the methods of choice for detecting low copy sequences in intact tissue samples.