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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
Ernst J. M. Speel, Frans C. S. Ramaekers, and Anton H. N. Hopman
1. Introduction
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. 1). 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
(2). 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 (3–5). Hence, in the end of the 1980s and
1990s, several strategies had been developed to improve the threshold levels as well as
the efficiency of nucleic acid detection in situ, such as target and signal amplification
methods (Table 1). In this chapter, we focus on the technical aspects of the primed in
situ labeling (PRINS) and in situ PCR procedures, originally introduced by Koch et al.
(6) and Haase et al. (7), respectively, as examples of nucleic acid target amplification
methods. The pros and cons of both techniques will be discussed and compared with
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
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