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

PRINS and In Situ PCR 407
Fig. 1. Basic differences between in ISH, PRINS, and cycling PRINS DNA labeling and
direct and indirect in situ PCR. *Incorporated reporter molecule (fluorochrome or hapten [biotin,
digoxigenin, dinitrophenyl]), which can be detected as described in Subheading 2.1.
digoxigenin, dinitrophenyl) nucleotides can be visualized by the additional application
of fluorochrome- or enzyme-conjugated avidin or antibody molecules, followed by
fluorescence microscopy or brightfield visualization of enzyme reaction products
(2). Image recording and analysis is usually performed by using a CCD camera or
confocal scanning laser microscope. In this overview, the use of radioactivity as reporter
molecule will not be considered because of disadvantages related to cost, instability,
biohazard potential, the time required for autoradiographic detection, and the poor
resolution of the final in situ signals.
2.2. Sample Fixation and Processing
In methanolacetic acid (31)-fixed metaphase spreads (adhered to noncoated glass
slides), a PRINS reaction with oligonucleotides specific for centromeric or telomeric
repeats usually runs for only 5 to 30 min, resulting in bright and easily localized signals
and a high signal-to-noise ratio (Fig. 2A).
Nevertheless, background staining might occur because of nicks in the chromosomal
DNA that may act as primers for unspecific labeling. Thus, it is recommended to
use freshly prepared chromosome preparations or optionally perform a DNA ligase
reaction to close the nicks before PRINS labeling (17,20). Also, in interphase cells
and frozen tissue sections, these repetitive target sequences can be efficiently detected
provided that the specimens are adhered to coated (e.g., organosilane) glass slides,
fixed in methanolacetic acid (31), and pretreated with a mild protease digestion
(e.g., 100 µg/mL pepsin or 0.025% proteinase K; Fig. 2B and refs. 12,18). Because
of the thorough fixation, short denaturation temperature, and speed of the reaction, the
morphology of chromosomes and cell nuclei in methanolacetic acid (31)-fixed cell
preparations is usually well preserved. On frozen and formaldehyde-fixed, paraffin-