John M. S. Bartlett.pdf - Bio-Nica.info

414 Speel, Ramaekers, and Hopman
technique has also been applied to identify endogenous DNA and RNA sequences in
human cells (for reviews, see refs. 8–10,13). In situ PCR techniques are theoretically
straightforward and comprise (1) sample fixation and pretreatment to improve the
accessibility of the target nucleic acid sequences by the PCR primers, nucleotides, and
Taq polymerase enzymes and to avoid diffusion of PCR-generated amplificants; (2)
PCR amplification in the cell by Taq polymerase using the target DNA as a template; and
(3) direct (by incorporation of labeled nucleotides during PCR) or indirect (by ISH
with labeled nucleic acid probes) detection of the amplified nucleic acid molecules.
Visualization of labeled target or probe DNA can be performed as described in
Subheading 2.1. Thus, in principle direct in situ PCR is identical to the cycling PRINS
procedure described above when cellular DNA is directly labeled during PCR cycling
(Fig. 1). Consequently, the conditions for optimal chromosome labeling by (cycling)
PRINS will also apply for the direct in situ PCR approach, although they need to be
adapted for the specimen of interest and the fixative used to process cells and tissues.
3.2. Sample Fixation and Processing
It has been reported that 4 to 10% buffered formaldehyde is the fixative of choice for
successful in situ PCR on cells and (frozen) tissues and that these specimens should be
adhered to coated (e.g., organosilane) glass slides to prevent loss of tissue adherence
during the in situ PCR procedure (5,9). In addition, cell and tissue preparations need
to be subjected to a protease (pepsin, trypsin, or protease K) treatment to create holes
in cellular membranes and remove cross-linked DNA-binding proteins from nuclear
DNA, thereby facilitating the accessibility of the nucleic acids in situ by the PCR and
detection reagents, as well as the ISH probes (in the indirect in situ PCR procedure).
Importantly, for every protease and tissue, the optimal balance between time and
concentration should be determined (usually 5 to 30 min of 2 mg/mL protease at room
temperature or 37°C) to avoid overdigestion, leading to poor tissue morphology and
possible leakage (diffusion) of amplified products from the cell in which they were
generated, or insufficient protein removal, resulting in a decreased or completely absent
in situ signal caused by very inefficient or failure of amplification (5,9). A striking
point to notice here is that the generally used protease pretreatment for in situ PCR
is less powerful than the one applied usually for optimal ISH on formaldehyde-fixed,
paraffin-embedded preparations, in which prior to the protease digestion treatments with
1 M sodium thiocyanate and, optionally, 85% formic acid/0.3% H 2 O 2 are performed
to achieve optimal conditions for ISH while preserving nuclear morphology (44).
Although this may still be sufficient to allow the PCR reagents to reach the target DNA,
we know from our own results that efficient in situ primer extension rather requires a
more powerful tissue pretreatment step than used for ISH (see Subheading 2.2. and
3.3.2.). Indeed, we have not been able to detect human centromere repeats by PRINS
labeling using tissue pretreatment conditions optimal for ISH with centromere-specific
plasmid probes (unpublished results), indicating that in situ primer extension during the
first couple of cycles of PCR on the slide is most likely impossible or very inefficient.
This might in part explain the low amplification efficiency (restricted sensitivity)
often obtained by in situ PCR, which furthermore can be caused by diffusion of the
synthesized DNA amplificants outside the cell (see Fig. 1 and below).