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

406 Speel, Ramaekers, and Hopman
Table 1
Approaches to Amplify Nucleic Acid Target Sequences and (Immuno)
Cytochemical Detection Signals in situ (Adapted from ref. 2)
Target
Reference
Nucleic acid target amplification
In situ polymerase chain reaction (in situ PCR) DNA (This chapter)
Primed in situ labeling (PRINS) DNA (This chapter)
and repeated /cycling PRINS
Rolling circle amplification Hybridized (55)
oligonucleotide
(Padlock probe)
In situ reverse transcriptase (RT) PCR RNA (8–10,13)
In situ self-sustained sequence replication (3SR) RNA (56)
In situ transcription /PRINS RNA (57,58)
Detection signal amplification
Branched DNA amplification (59)
Catalyzed reporter deposition /tyramide signal amplification (CARD/ TSA) (2,14)
Mirror image complementary antibodies (MICA) (60)
Enzyme antibody polymer system (EPOS/EnVision) (61,62)
Enzyme-labeled antibody–avidin conjugates (63)
End Product Amplification (anti-DAB antibody strategy) (64)
the current protocols for ISH using tyramide signal amplification. Special emphasis
will be on the conditions needed to achieve an optimal balance between nucleic acid
detection in situ and preservation of cell and tissue morphology, including discussions
on sample fixation and pretreatment, oligonucleotide/probe hybridization, in situ
primer extension and amplification, and detection of incorporated reporter molecules.
For more comprehensive reviews and applications of PRINS, in situ PCR, and ISH,
we refer to the literature (2,8–17).
2. PRINS
2.1. PRINS vs ISH
Particularly in the field of cytogenetics, the PRINS labeling technique has become an
alternative to ISH for the localization of nucleic acid sequences in chromosome and cell
preparations (6,8,12,17). Occasionally, its application to the detection of chromosome
copy numbers and viral DNA in frozen and formaldehyde-fixed, paraffin-embedded
tissue sections have been reported (12,18,19). Whereas in an ISH approach a nucleic
acid probe with incorporated reporter molecules is hybridized to its cellular target, the
PRINS method is based on the use of high concentrations of unlabeled primers (restriction
fragment, PCR product, or oligonucleotide) that allow a very fast hybridization
(annealing) to denatured, complementary target sequences in situ (Fig. 1).
These primers serve as initiation sites for in situ chain elongation catalyzed by
Taq DNA polymerase (in the appropriate buffer containing 1.5 mM MgCl 2 ) using the
target DNA as a template. DNA labeling takes place during the elongation step
when labeled nucleotides are incorporated. Fluorochrome-labeled nucleotides can
be detected directly by fluorescence microscopy, while haptenized (e.g., biotin,