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

410 Speel, Ramaekers, and Hopman
polymerase (31,32). In principle, the number of in situ DNA targets to be detected
simultaneously can be extended further by increasing the number of subsequent PRINS
reactions applying different reporters/fluorochromes or combinations of two or more
reporters in different ratios. However, the efficiency of this procedure is expected to
decrease after multiple sequential PRINS reactions and, therefore, ISH is the preferred
technique to use for the localization of more than 3 targets (Table 2 and refs. 34,35).
PRINS has also been successfully combined with the immunocytochemical detection
of proteins in multicolor approaches to, for example, immunophenotype cells harboring
a specific chromosomal aberration or viral infection, to investigate chromosome
distribution and segregation in cells during processes such as polyploidization and
aneuploidization, and to identify possible relationships of different families of DNA
sequences with, for example, proteins associated with different chromosome-specific
structures, such as the kinetochore complex (see Chapter 63).
Particularly, the rapidity, improved probe accessibility and lack of formamide for
hybridization, thereby preventing the destruction of protein epitopes, are advantages of
applying PRINS instead of ISH in these procedures.
2.5. Improvement of the Detection Sensitivity
The major drawback of PRINS for a long time proved to be its inability to convincingly
detect single-copy gene sequences (27). This is caused by the fact that the in situ
primer extension by Taq DNA polymerase in the biological material (adhered to glass
slides) is limited to relatively short lengths (in the range of maximum a few hundreds
of basepairs), probably caused by (1) the local chromatin organization of the target
sequence; (2) the binding of (part of) the target to remnant proteins or the glass slide;
and/or (3) the presence of nicks in the DNA where the polymerase reaction will stop
(12). As a consequence, a single PRINS reaction to localize a single copy gene sequence
with one primer (pair) will hardly result in a positive signal in the microscope, as
the current detection sensitivity with ISH is approx 1 to 5 kb (2,36). The problem
has now been overcome by using either multiple target-specific primers in a single
PRINS reaction combined with reporter detection by the tyramide signal amplification
procedure (28) or repeated PRINS reactions with the same reaction mixture and primers
on specimen preparations (also called cycling PRINS) (8,20,29,37,38 and Chapter 60).
Essential improvements of the first approach consisted of (1) the treatment of 1-d-old
metaphase slides with 0.02 N HCl to remove loosely bound protein and thereby to
render the DNA more accessible to the primer; (2) the use of multiple (four to five)
primers for one locus; (3) one PRINS reaction and stringent washings in SSC to achieve
optimal specificity; (4) the use of TaqStart, a monoclonal antibody against Taq DNA
polymerase, which prevents nonspecific amplification and formation of primer-dimers;
and (5) the use of biotin incorporation combined with biotin-labeled tyramide signal
amplification (28). With this procedure a couple of single-copy genes have been
detected in chromosome preparations with high efficiency (39). It will be interesting
to see whether this approach can be applied to clinical cell and tissue specimens as
well, for example, for rapid and reliable detection of microorganisms and chromosomal
alterations in cancer specimens. Alternatively, cycling PRINS has been used on
metaphase spreads and blood smears to detect low and single-copy DNA sequences
resulting in more intense (up to 15×) fluorescence in situ signals than seen after a single