In situ polymerase chain reaction: general methodology and recent advances.
In situ PCR is a new molecular technique, that combines the extreme sensitivity of PCR with the cellular localization provided by in situ hybridization (ISH), through the amplification of specific gene sequences within intact cells or tissue sections and increasing copy numbers to levels detectable by ISH or immunohistochemistry. In addition to the detection of viral DNA (CMV, HBV, HIV), we have used this technique for the study of DNA rearrangements, chromosomal translocations (t14;18) and viral RNA (HCV) in cells in suspension, cytocentrifuge preparations or archival tissue sections. We compared different approaches to in situ amplification of target sequences and visualization of PCR products by either subsequent ISH (indirect in situ PCR) or by direct detection of labeled nucleotides, which have been incorporated during PCR (direct in situ PCR). Our results indicate, that in situ PCR includes a number of different techniques, which are not equally applicable to all types of samples. In situ PCR appears to be most effective for the detection of DNA in single cell preparations with controlled fixation and pretreatment, although the quantification of results remains problematic. Artifacts caused by diffusion and extracellular generation of PCR products are a significant problem potentially leading to false positive results. In situ PCR works less efficiently in archival tissue sections due to poor quality of nucleic acids and retention of PCR products. Direct in situ PCR yields less specific results than indirect in situ PCR and requires additional controls such as omission of primers in the reaction mixture to detect artifacts.(ABSTRACT TRUNCATED AT 250 WORDS)
Komminoth
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Abteilung für Zell- und Molekularpathologie, Universität Zürich, Schweiz.
Heitz
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PU
Long
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D016477:Artifacts; D002874:Chromosome Mapping; D002877:Chromosomes, Human; D004247:DNA / Q000032:analysis* / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis*; D005188:False Negative Reactions; D005189:False Positive Reactions; D015321:Gene Rearrangement; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D017403:In Situ Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000032:analysis* / Q000235:genetics; D015203:Reproducibility of Results; D014178:Translocation, Genetic; D014777:Virus Diseases / Q000175:diagnosis* / Q000473:pathology*
D004279:DNA, Viral; D012313:RNA; D004247:DNA