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

PRINS and In Situ PCR 411
PRINS reaction (Fig. 1, refs. 20,29,37,38,40). The repeated PRINS reactions appear
to label not only the target sequence (as observed after the last PRINS cycle) but also
the DNA that has been synthesized during the preceding cycles. Several protocols have
been described with suggestions for optimal gene localization, including (1) the use
of single or multiple primers generating DNA products of 250 to 550 bp in length; (2)
alternative fixation and pretreatment protocols (e.g., ethanol and microwave treatment
instead of methanol:acetic acid [31] fixation); 3) the use of a hot start (applying the
reaction mixture and/or Taq polymerase at the annealing or a higher temperature to
the slide to avoid mispriming or primer oligomerization during PRINS/PCR) before 1
to 20 PRINS cycles, eventually preceding by PCR with the same primers but without
labeled nucleotides; and (4) adaptation of the PRINS reaction volume and conditions
on the glass slide during cycling. However, despite these recommendations, a main
concern comprises the reproducibility of cycling PRINS, because in most studies
variable and relatively low frequencies (10–70%) of chromosomes or cells harboring
PRINS signals were reported. Besides the discussed possibilities that may result in
background (nicks, mispriming), the major disadvantage of applying multiple cycles
of PRINS (or PCR, see later) on cell and tissue preparations is the inevitable diffusion
of newly synthesized DNA products, inextricably bound up with the denaturation
steps, from the site of synthesis inside and/or outside the cells, followed by possible
extracellular generation of amplificants (Fig. 1. refs. 9,20,29,41–43). Several studies
have provided evidence for this phenomenon by demonstrating the expected DNA
products using gel electrophoresis of the reaction mixture after cycling PRINS. The
observation of stronger but often more diffuse and less discretely localized PRINS
signals in only a low percentage of chromosomes or cell nuclei fits in with the
view that labeled DNA in these cases is retained at or near the site of synthesis by
possible entrapment in the chromatin or nonspecific binding to the surrounding cellular
structures, whereas diffusion of DNA away from the site of synthesis has been taken
place in the remaining negative chromosomes or nuclei. That diffusion occurs may be
explained by the fact that methanolacetic acid (31) fixation is an effective procedure
to extract proteins, thus limiting the possibillities to entrap synthesized DNA molecules
efficiently in the chromosome structures. Although postfixation in paraformaldehyde
after cycling PRINS has been suggested to reduce diffusion of produced DNA (17), this
will of course only be of help in the chromosomes and nuclei harboring signals.
Thus, because of several drawbacks concerning the efficiency and reproducibility of
cycling PRINS in localizing either low or single-copy DNA sequences in situ, a single
PRINS reaction with multiple primers combined with tyramide signal amplification
is recommended for this purpose (as a rapid alternative to ISH with locus-specific
probes).
3. In Situ PCR
3.1. In Situ PCR vs Cycling PRINS
In line with the development of PRINS, several groups working in the fields of
pathology and microbiology have introduced the successful combination of PCR and
ISH to visualize specific amplified nucleic acid sequences in cell and particularly
formaldehyde-fixed, paraffin-embedded tissue preparations. Most studies have focused
on the detection of (pro)viral (foreign) nucleic acid sequences, but in addition the