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

PRINS and In Situ PCR 409
embedded tissue sections, however, nuclear morphology may be less well preserved
after PRINS DNA labeling because in our experience more powerful tissue pretreatment
steps (extensive protein removal) are required than used for ISH to allow for an
efficient in situ primer elongation step (18). In combination with the high denaturation
temperature, as a result, discrete nuclear morphology is more difficult to maintain.
Although the small size of oligonucleotides used for PRINS (usually 18 to 35 nucleotides)
greatly facilitates their accessibility to genomic target sequences in comparison
with the larger probes used for ISH, the subsequent in situ primer elongation reaction,
thus, appears to be the success-determining step in the PRINS procedure. Particularly
on tissue sections, this requires more extensive pretreatment steps than necessary for
optimal ISH, which is a perhaps unexpected but important finding to realize.
2.3. Primers and Hybridization Conditions
The specificity of the PRINS reaction is dependent on the choice of the primer
sequences as well as the conditions of primer annealing and extension used. Both single
and paired primers have been described for use in PRINS. On basis of interchromosomal
differences in the α-satellite and other satellite DNA repeats, human chromosomespecific
PRINS primers have been designed for all human chromosomes, except for
chromosome 14 and 22, which are detected simultaneously with a single 14/22 primer
(21,22). Chromosome-specific PRINS primers have also been constructed for other
species, such as the pig (23), as well as for other human DNA repeat regions, including
telomeres and ALU sequences (24–26) and for specific single-copy genes (27,28).
Optimization of the stringency of primer annealing (in sufficient reaction volume to
prevent evaporation leading to concentration and temperature shifts), which reduces
mispriming during the PRINS reaction, has been established by substituting the initially
used thermoblocks or waterbaths by programmable thermal cyclers equipped with a
flat plate block, which allow for precise and durable temperature control (up to 0.2°C
accuracy). As a consequence, semi-automated PRINS protocols are now available,
which offer a high reproducibility of nucleic acid labeling (8,9,29,30).
2.4. Multicolor PRINS and Combination with Immunocytochemistry
Multicolor detection of up to three DNA target sequences in situ have been performed
using subsequent PRINS reactions with different primers and labeled oligonucleotides
(30–33). This enables several targets to be analyzed simultaneously (Fig. 2C,D),
which, for example, can make the evaluation of chromosome aberrations in clinical
samples more robust. In addition, multicolor PRINS might be especially valuable in
cases where only one or a few cells are available for analysis, for example, preimplantation
genetic diagnosis. A prerequisite of performing multiple PRINS reactions in
sequence is to stop the first PRINS reaction adequately and to avoid the further labeling
of the first produced DNA strand with differently labeled nucleotides used in the
subsequent PRINS reaction. This can be achieved by incubation with ddNTPs and
Klenow DNA polymerase to block the free 3′ ends of the produced DNA strand (16,33),
although others have reported that this step can be omitted, probably because of either
complex chromatin conformations or incomplete DNA denaturation after relatively
long extension reactions in the first PRINS reaction that hamper the access of Taq