Summary
PCR in situ promises the ability to amplify and detect very low levels of target nucleic acid in tissues. Despite considerable effort, the technique is still technically difficult and has not yet proved to be reliable or reproducible. We have now identified a number of factors which can contribute to the poor amplification of the target DNA and to the generation of false-positive signals. These factors include the effects of fixation; reagent abstraction, DNA degradation, DNA end-labelling and product diffusion. We present evidence to show that formaldehyde fixation cross-links histones to DNA and thus restricts the subsequent amplification of target sequences by PCR. End-labelling of DNA occurs when direct incorporation is used to detect amplified products and this gives rise to false-positive signals. Amplified products can also diffuse out of cells and into neighbouring cells which do not contain target sequences. They can undergo re-amplification within these cells giving rise to false-positive signals. We believe considerable caution should be exercised in the interpretation of results generated using PCR in situ.
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References
bagasara, o., hauptman, s. p., lischner, h. w., sachs, m. & pomerantz, r. j. (1992) The detection by in-situ polymerase chain reaction of provirus in mononuclear cells of certain individuals infected with HIV-1 N. Engl. J. Med. 326, 1385–91.
bagasara, o., seshamma, t., oakes, j.w. & pomerantz, r.j. (1993) High percentage of CD4 positive lymphocytes harbour the HIV-provirus in the blood of certain infected individuals. AIDS 7, 1419–25.
braga, v.m.m. & gendler, s.j. (1994) Co-amplification of two cDNAs in RT-PCR can alter the yield of both products. Biotechniques 17, 228–30.
chiu, k.p., cohen, s.h., morris, d.w. & jordan, g.w. (1992) Intracellular amplification of proviral DNA in tissue sections using the polymerase chain reaction. J. Histochem. Cytochem 40, 333–41.
cox, m. m. & lehman, i. r. (1981) Renaturation of DNA: a novel reaction of histones. Nucleic Acids Res. 9, 389–400.
crisan, d. & mattson, j. c. (1993) Retrospective DNA analysis using fixed tissue specimens. DNA Cell Biol. 12, 455–64.
doenecke, d. (1978) Digestion of chromosomal proteins in formaldehyde treated chromatin. Hoppe Seylers Z Physiol Chem 359, 1343–52.
folks, t. m., powell, d., lightfoote, m., koenig, s., fauci, a. s., benn, s., rabson, a., daugherty, d., gendelman, h. e., hoggan, m. d., venkatesan, s. & martin, m. (1986) Biological and biochemical characterization of a cloned Leu 3 - cell surviving infection with the acquired immune deficiency syndrome retrovirus. J. Exp. Med. 164, 280–90.
forsthoefal, k.f., papp, a.c., snyder, p.j. & prio, t.w. (1992) Optimization of DNA extraction from formalin fixed tissue and its clinical application in Duchenne muscular dystrophy. Am. J. Clin. Pathol. 98, 98–104.
gallo, r. c., salahuddin, s. z., popovic, m., sheareer, g. m., kaplan, m., haynes, b. f., palker, t. j., redfield, r., oleske, j., safai, b., White, g., foster, p. & markam, p. d. (1984) Frequent detection and isolation of cytopathic retrovirus HTLV-III from patients with AIDS and at risk for AIDS. Science 224, 500–3.
greer, c. e., peterson, s. l., kiviat, n. b. & manos, m. m. (1991a) PCR amplification from paraffin-embedded tissues. Effects of fixative and fixation time. Am. J. Clin. Pathol. 95, 117–24.
greer, c. e., lund, j. k. & manos, m. m. (1991b) PCR amplification from paraffin-embedded tissues: recommendations on fixatives for long-term storage and prospective studies. PCR Meth. Appl. 1, 46–50.
haase, a. t., retzel, e. f. & staskus, k. a. (1990) Amplification and detection of lentiviral DNA inside cells. Proc. Natl. Acad. Sci. USA 87, 4971–5.
hahn, b. h., shaw, g. m., arya, s. k., popovic, m., gallo, r. c. & Wong-Staal, f. (1984) Molecular cloning and characterization of the HTLV-III virus associated with AIDS. Nature 312, 166–9.
jackson, d. p., lewis, f. a., taylor, g. r., boylston, a. w. & quirke, p. (1990) Tissue extraction of DNA and RNA and analysis by the polymerase chain reaction J. Clin. Pathol. 43, 499–504.
komminoth, p. & long, a. a. (1993) In-Situ polymerase chain reaction. An overview of methods, applications and limitations of a new molecular technique. Virchows Arch B Cell Pathol. 64, 67–73.
komminoth, p., long, a.a., ray, r. & Wolfe, h. j. (1992) In situ polymerase chain reaction detection of viral DNA, single-copy genes, and gene rearrangements in cell suspensions and cytospins. Diagn. Mol. Pathol. 1, 85–97.
long, a. a., komminoth, p., lee, e. & Wolfe, h. j. (1993) Comparison of indirect and direct in-situ polymerase chain reaction in cell preparations and tissue sections. Histochemistry 99, 151–62.
morikawa, s., tatsumi, e., baba, m., harada, t. & kimio, y. (1978) Two E. rossette-forming lymphoid cell lines. Int. J. Cancer 21, 166–70.
nuovo, g. j. (1992) PCR in-situ Hybridization, Protocols and Applications. New York: Raven Press.
nuovo, g. j., gallery, f. & macconnell, p. (1992) Detection of amplified HPV 6 and 11 DNA in vulvar lesions by hot start PCR in situ hybridization. Mod. Pathol. 5, 444–8.
nuovo, g. j., gallery, f., macconnell, p. & bloch, w. (1993) Importance of different variables for enhancing in situ detection of PCR-amplified DNA. PCR Meth. Appl. 2, 305–12.
nuovo, g. j., becker, j., simsir, a., margiotta, m., khalife, g. & shevchuk, m. (1994a) HIV-1 nucleic acids localize to the spermatogonia and their progeny. A study by polymerase chain reaction in-situ hybridization. Am. J. Pathol. 144, 1142–8.
nuovo, g. j., gallery, f., macconnell, p. & braun, a. (1994b) In-situ detection of polymerase chain reaction amplified HIV-1 nucleic acids and tumour necrosis factor - α RNA in the central nervous system. Am. J. Pathol. 144, 659–66.
ou, c. y., kwok, s., mitchell, s. w., mack, d. h., sninsky, j. j., krebs, j. w., feorino, p., Warfield, d. & schokhetman, g. (1988) DNA amplification for direct detection of HIV-1 in DNA of peripheral blood mononuclear cells. Science 239, 295–7.
popovic, m., sarngadharan, m. g., read, e. & gallo, r. c. (1984) Detection, isolation and continuous production of cytopathic retrovirus HTLV-III from patients with AIDS and pre-AIDS. Science 224, 497–500.
sallstrom, j. f., zehbe, i., alemi, m. & Wilander, e. (1993) Pitfalls of in-situ polymerase chain reaction (PCR) using direct incorporation of labelled nucleotides. Anticancer Res 13, 1153.
simmonds, p., balfe, p., peutherer, j. f., ludlum, a., bishop, o. & leigh Brown, a. (1990) Human immunodeficiency virus infected individuals contain provirus in small numbers of peripheral mononuclear cells and at low copy numbers. J. Virol. 64, 864–72.
sodrowski, j. g., rosen, c. a. & haseltine, w. a. (1984) Trans activating transcriptional activation of the long terminal repeat of human T lymphotropic viruses in infected cells. Science 225, 381–5.
solomon, m. j. & varshavsky, a. (1985) Formaldehyde-mediated DNA-protein crosslinking: a probe for in-vivo chromatin structures. Proc. Natl. Acad. Sci. USA 82, 6470–4.
staskus, k. a., couch, l., bitterman, p., retzel, e. f., zupancic, m., list, j. & haase, a. t. (1991) In situ amplification of visna virus DNA in tissue sections reveals a reservoir of latently infected cells. Microb. Pathog. 11, 67–76.
teo, I. A. & shaunak, S. (1995) PCR in situ: an appraisal of an emerging technique. Histochem. J. (in press).
tokuda, y., nakamura, t., satonaka, k., maeda, s., doi, k., baba, s. & sugiyama, t. (1990) Fundamental study on the mechanism of DNA degradation in tissues fixed in formaldehyde. J. Clin. Pathol. 43, 748–51.
yap, e. p. & mcgee, j. o. (1991) Slide PCR: DNA amplification from cell samples on microscopic glass slides. Nucleic Acids Res. 19, 4294.
zaki, s. r., heneire, w., lotheld, l. m., greer, p. w., sinha, s. d. & folks, t. m. (1994) In-situ polymerase chain reaction amplification: applications and current limitations. AIDS 8, 1186–7.
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Teo, I.A., Shaunak, S. PCR in situ: aspects which reduce amplification and generate false-positive results. Histochem J 27, 660–669 (1995). https://doi.org/10.1007/BF00216679
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DOI: https://doi.org/10.1007/BF00216679