The In Situ PCR:Amplification and Detection in a Cellular Context

Ernest F. Retzel, Katherine A. Staskus, Janet E. Embretson and Ashley T. Haase Department of Microbiology, University of Minnesota, Minneapolis, MN 55455 Copyright ?1994 The Regents of the University of Minnesota Introduction The diversity of pathogenesis presents a spectrum of challenges to the researcher, who is required to utilize a wide variety of tools and techniques, including those of a histological, immunological and molecular nature. Within the larger context of pathogenesis, the determination of the mechanism of viral latency and slowly progressing viral diseases is particularly provoking. Here, the molecules involved in the initiation and progression of disease may be present in vanishingly small quantities in a minor population of cells or tissues, and the dynamics of expression of functions within the cells will vary over time. In many of these slowly-evolving diseases, requiring months or years to manifest clinically, it has been shown that, with respect to viral genetic information, the majority of the infected or affected population is in a transcriptionally inactive state, and at a level of one genome [or gene] per host cell.

An example of one of the most difficult issues in viral pathogenesis today is that presented by the lentiviruses, members of the "complex" subgroup of the retroviruses, which includes HIV, the causative agent of AIDS in humans, and the prototype for the class, visna-maedi, which causes neurological and pulmonary disease in sheep. Upon infection with these agents, the provirus frequently integrates into the host genome and establishes a persistent infection, whereby the infected cells are characterized by, among other things, a transcriptionally-quiescent state with respect to the viral antigens, which allows the infected cells to escape host immune surveillance [the Trojan horse mechanism]. It is this state which is pathogenically and epidemiologically most interesting, and potentially the most lethal, since these cells provide a reservoir for the future release of active virus. It is these latently infected individual cells, then, which need to be discovered and enumerated in order to gain some insight into the essence and the extent of the infection, the progression to expression and, eminently, the control of this progression.

The techniques of nucleic acid hybridization and the polymerase chain reaction [PCR] have been used extensively to investigate these issues of pathogenesis. While powerful in their own right, these techniques are essentially population studies: nucleic acids are isolated from a population of cells which contains either a sufficient number of molecules to detect directly by standard hybridization techniques, or, when a subpopulation contains as little as a single copy of nucleic acid, that molecule amplified by the PCR, and detected after amplification. In situ hybridization applies and extrapolates the technology of nucleic acid hybridization to the single cell level, and, in combination with the artistry of cytochemistry and immunocytochemistry, permits the maintenance of morphology and the identification of cellular markers to be maintained and identified, allows the localization of sequences to specific cells within populations, such as tissues and blood samples. However, with in situ, the technology is limited primarily to the detection of non-genomic material [e.g., RNA], reiterated genes or multiple genomes, since the limits of detection in most situations are several copies of the target nucleic acid per cell. In part due to these copy number considerations, hybridizations for RNA are considerably more sensitive than for DNA detection. In addition, other factors which affect the sensitivity of the sensitivity of the technique toward RNA targets are the strandedness of the target molecule and the lack of a complementary sequence proximal to the target sequences, which kinetic theory would predict to be a preferential substrate for annealing. Other techniques, specifically the reverse transcriptase-catalyzed in situ transcription, have been used as well to detect RNAs which occur at relatively high copy number. The single-copy problem proffered by the slow disease agents referred to above, however, is beyond the realm of repeatable reality with conventional in situ techniques.

Since the viral nucleic acid within these infected cells is below the level of routine detection, the application of amplification technologies, of which the PCR is the most logical choice due to its exquisite sensitivity, is essential. Clearly, the solution-phase corollary of this problem, namely the amplification and detection of DNA purified from a single cell, either as an individual cell or within a population of dissimilar cells, has been resolved [see Chapter xy]. For the histological identification of that cell within a population, such as in a tissue section or within a blood sample, the problem becomes more complex. In addition to the standard reaction components, consisting of enzyme, target DNA, substrate and primers, which must be optimized in addition to varying cofactors [Mg, primer-template ratios, etc.], one must consider other variables and potential problems, all of which have direct consequences in the success of this technique. The importance of the preservation of morphology and antigens of cells and tissues limits the choice of fixation methods; these procedures are crucial to the detection of sequences generally in in situ hybridization, and are even more so when considering the PCR in situ . The modified cellular milieu, maintained as a consequence of the fixation procedure, dramatically affects the efficiency of amplification, as it introduces potential interference from protein contaminants and cross-linking of nucleic acids and proteins. This is compounded by the general reduction of signal that occurs in solid-phase systems. The diffusion of reaction components into the cells likely also proceeds at a lower rate than in solution. Finally, the amplified product DNA must be retained within the permeabilized cell for the detection by radiolabelled probes; this necessitates a relatively large size for the designed product DNA, which is contrary to the relatively low efficiency for the in situ amplification reaction. All of these considerations amplify the overall problem of detecting PCR-generated sequences within a morphologically-identifiable cell, in addition to amplifying the sequences.