PCR-based detection of nucleic acids in chromosomes, cells, and tissues: technical considerations on PRINS and in situ PCR and comparison with in situ hybridization.

Abstract

The polymerase chain reaction (PCR) is an extremely sensitive technique allowing the detection of rare and low copy nucleic acid sequences (up to 1–10 copies in DNA or mRNA extracts from 1 million cells) by solution-phase amplification using specific primer sets and Taq DNA polymerase and visualization of the resulting PCR products by gel electrophoresis and blotting techniques (see chapters in this book). However, the obligatory cell and tissue destruction required for nucleic acid extraction does not permit the correlation of results with histopathological features or the localization of targets in specific cell types. This may now be overcome by combining recently developed micromanipulation systems, such as laser-assisted microdissection, to isolate the cells of interest (up to the level of a single cell) from a large population of cells or from the tissue, and to apply single-cell PCR on the extracted nucleic acids (for a review, see ref. ). Alternatively, and in case it is unknown in which cell a certain target nucleic acid, for example, a virus particle, may be present, cellular localization of DNA and RNA can be accomplished by in situ hybridization (ISH). This procedure has a history of more than 30 years and has been improved continuously. In particular, the development of nonradioactive approaches and the recent implementation of tyramide signal amplification have made ISH a powerful technique for use in many applications (). Some 10 to 15 years ago, however, ISH detection limits were only in the range of 10 to 20 copies of mRNA or viral DNA per cell, and probe detection periods could be very long when using radioactive procedures (, , ).