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

PRINS and In Situ PCR 415
3.3. In Situ Amplification and Detection of Intracellular PCR Products
PCR protocols with optimal stringency of primer annealing and using generally
longer extension times, increasing concentrations of Taq polymerase and MgCl 2 , and /or
the addition of bovine serum albumin in the reaction mixture on the cell and tissue
preparations as compared with solution-phase PCR have been established by using
the newly designed programmable thermal cyclers as described for (cycling) PRINS
(see Subheading 2.3. and refs. 5,9,30). After PCR amplification, visualization of
intracellular PCR products is achieved either directly through immunohistochemical
detection of labeled nucleotides (see Subheading 2.1.) that have been incorporated
into PCR products during thermal cycling (direct in situ PCR) or indirectly by ISH
with a labeled probe and subsequent immunohistochemical detection (see Chapter
63 and refs. 5,9).
3.3.1 Direct In Situ PCR
Although direct in situ PCR is more rapid than indirect in situ PCR by eliminating
the need for subsequent ISH, this procedure has proved unreliable with respect to
the specificity of the results obtained (9,43,45). Even when the hot start procedure
is performed (see Subheading 2.4.), the direct detection approach yields significant
false-positive results, especially when working with tissue sections that have been dried
at 56 to 65°C for several hours (introduction of nicks in the DNA) (5,9). This is the
result of a number of artifacts, including incorporation of labeled nucleotides into (1)
nonspecific PCR products resulting from mispriming (“endogenous priming” artifacts)
and primer oligomerization, which seems to occur less likely inside nuclei (5) but may
play a role during extracellular amplification of diffused DNA products); (2) singleand
double-stranded nicks introduced by tissue fixation, cutting, and drying; and (3)
fragmented DNA undergoing “repair” by DNA polymerase (“repair” artifacts). Repair
artifacts may particularly be evident in apoptotic cells or samples that have been
pretreated with DNase before in situ reverse transcriptase PCR for mRNA detection
(9,45). These artifacts may only slightly be reduced by using an exonuclease-free
DNA polymerase or by carrying out a DNA ligase reaction to close the nicks or a
ddNTP reaction to prevent a subsequent extension reaction (9,17,33). Thus, caution
and adequate use of appropriate controls are recommended in the interpretation of data
produced by direct in situ PCR (Table 3 and Subheading 3.4.).
3.3.2. Indirect In Situ PCR
The indirect in situ PCR technique, therefore, is the preferred approach to use,
because the intracellular target-specific PCR products are identified by ISH, whereas
the nonspecific amplificants will not be detected. Probes targeted to regions in between
the primers used for PCR, usually oligonucleotide probes of 20 to 40 bases, represent
the ideal ISH probes for reasons of specificity because they assure that the detected
signal is the PCR product and not the result of for example primer oligomerization. ISH
with these small probes, however, is usually less sensitive than with cDNA or genomic
probes. Because primer oligomerization seems not to occur inside nuclei during PCR
(10), the latter probes are recommended because of the increased number of reporter
molecules they carry, leading to a higher detection sensitivity. Immunohistochemical