Carlos A. Muro-Cacho, M.D., Ph.D.

Department of Pathology, H. Lee Moffitt Cancer Center and Research Institute and University of South Florida College of Medicine, Tampa, Florida, USA

Received 1/10/97 Accepted 7/15/97

7. APPLICATIONS

Despite technical difficulties, In situ PCR protocols are being utilized in an increasing number of applications. The technique has particular potential in areas such as embryogenesis, organogenesis, infectious diseases, genetics, immunology, neoplasia or pathology. New genes are continuously being added to the list of target sequences, detected by In situ PCR, that includes infectious agents such as human immunodeficiency virus (HIV-1), simian immunodeficiency virus (SIV), human papilloma virus (HPV), hepatitis B virus (HBV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpes virus-6 (HHV-6) and herpes simplex virus (HSV), and molecules such as p53, surfactant protein A, estrogen receptor, growth factors, growth factor receptors, transferrin, and adrenomedullin (88-110). Almost every tissue has been successfully tested (3, 48-57).

One of the areas where In situ PCR methodology has proven its value is the detection of viral sequences that are present in vanishingly small numbers (48-57). This is particularly true for slowly progressing viral diseases. Among these are the group of lentiviruses of which HIV is a member where In situ PCR has provided a ten-fold increase in sensitivity over the conventional techniques (51-53). Furthermore, the Creutzfeld-Jacob virus of Progressive Multifocal Leukoencephalopathy has been demonstrated by In situ PCR in an archival material from 1958, confirming previous solution PCR data and demonstrating that DNA sequences are preserved for long periods of time in routinely processed tissues (57). The increased sensitivity of In situ PCR offers the opportunity for viral latency studies, overcoming the need to activate the viruses in vitro prior to detection. For example, Herpes Simplex Virus was detected in the trigeminal ganglia of latently infected mice. In situ PCR revealed three times more infected neurons than it was shown previously (56).

A fast, alternative method to In situ Hybridization, Primed In situ DNA Synthesis (PRINS) (60, 61) involves annealing of oligonucleotides or short DNA fragments to denatured complementary nucleic acid sequences (i.e., metapahase or interphase chromosomes) on slides. A thermostable DNA polymerase incorporates nonradioactive labeled nucleotides that are detected by immunofluorescence. A variety of suitable primers for chromosomal sequences are available. A further increase in sensitivity has been achieved with Cycling PRINS. To detect low copy or unique sequences, the PRINS technique can be modified by subjecting the chromosomal preparation to amplification cycles that incorporate biotin or digoxigenin-labeled nucleotides. Detection can be accomplished by immunofluorescence with suitable antibodies.

A related technique, Self-Sustained Sequence Replication-Based Amplification (3SR) (63, 64) is a reiterated cycling of reverse transcription and amplification reactions, catalyzed by AMV-RT and T7 RNA polymerase, intended to replicate a RNA target via RNA/DNA hybrids and double-stranded cDNA intermediates. The cDNA copy of the original mRNA incorporates a T7 RNA polymerase promoter that is used as template for a 10 million fold amplification within 2 h. The destruction of RNA in RNA/DNA hybrids by E. coli RNAseH eliminates the need for denaturation steps.

Obstacles to the wide application of In situ PCR have been low amplification efficiency, poor reproducibility, and difficulty in quantitation (21). Improvements in the technology, however, are likely to reduce these drawbacks. Newer reported systems, such as the nanogold-silver detection (51) or the catalyzed reporter deposition methods (37) may reduce background. Another innovative procedure takes advantage of a fluorochrome-labeled oligonucleotide probe, specific for a region of the amplified DNA between the PCR primer sequences, that is designed to emit fluorescence signal only after hybridizing to the appropriate template (4,21).

It is expected that In situ PCR protocols will be further simplified in the near future and that this methodology and its variants, despite limitations, will find a deserved place in the repertoire of methods available to the researchers.