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

420 Speel, Ramaekers, and Hopman
advantages of PRINS over ISH, for example, the ability to discriminate between human
centromeres 13 and 21 for chromosome enumeration analysis in prenatal diagnosis,
have been counteracted by the development of specific probe sets that combine
chromosome-specific single-copy or chromosome painting probes for discrimination.
In methanolacetic acid (31)-fixed and protease-pretreated frozen tissue sections
and cell preparations (touch preparations, blood smears, smears of bone marrow
aspirates) so far only repetitive sequences have been visualized efficiently by a single
PRINS reaction (8,12,18). Single-copy DNA sequences have been successfully detected
by cycling PRINS and ISH (2,40), but because of the drawbacks involved with the
use of the cycling PRINS procedure as described above an ISH approach is preferred
for this purpose. The most optimal results have been obtained by applying (single
or multiple-target) ISH to 70% ethanol-fixed samples that have been pretreated with
pepsin (44,53).
A couple of studies have compared DNA detection, particularly genomic human
papillomavirus DNA, in formaldehyde-fixed, paraffin-embedded cell and tissue preparations
by in situ PCR, PRINS, and ISH (5,19,51,54). With all techniques HPV-specific
sequences were efficiently detected, but only the one or two HPV 16 DNA sequences,
known to be integrated in the genome of the human SiHa cell line, could be visualized by
ISH or indirect in situ PCR (see also Fig. 2E). Interestingly, two of these studies (51,54)
reported the use of ISH in combination with tyramide signal amplification as the method
of choice, since it provided the same sensitivity and a much better reproducibility and
reliability than the more cumbersome and poorly reproducible indirect in situ PCR
method. Apparently, under optimal conditions ISH combined with the high amplification
power of the signal amplification system can result in the same detection limits as
can be reached by the relatively low amplification efficiency of indirect in situ PCR
(as compared with solution-phase PCR). Furthermore, it provides optimal localization
of signals and discrimination between viral integration and replication within wellpreserved
cells and nuclei (Hopman et al., manuscript in preparation). This is almost
impossible to achieve with in situ PCR because of diffusion of PCR products in the cell
nucleus as well as leakage out of the nucleus to target-negative cells, which is still the
most important disadvantage of an in situ PCR approach.
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