40
Chromosome, Microdissection and Microcloning
layer of fluorochrome-coupled avidin. The
DNA to be used for
in situ
work should be
of the highest quality. The probe should
be devoid of repetitive sequences. Any
repeat sequences that are present must
be removed by subcloning into a suitable
vector or by using suppression hybridiza-
tion in the detection reaction. All other
components for
in situ
hybridization are
readily available in kits from biochemi-
cal companies specializing in molecular
biology products.
There are at least three methods for
the identiFcation of speciFc chromosomes
and chromosomal bands. GTG banding
followed by ±ISH and propidium iodide
fluorescence staining is commonly used.
In this case, ±ISH is performed accord-
ing to standard procedures, whereupon
photomicrographs from ±ISH and GTG
banding are compared to identify the
chromosome(s) harboring the gene of in-
terest. A second method involves ±ISH
hybridization followed by R-banding using
Hoechst dye. Chromosomes are stained
with Hoechst 33258 dye for 15 min after
±ISH, UV-irradiated for 20 min at 365 nm,
and then counterstained with propidium
iodide. ±ISH signals and R-banding using
propidium iodide or DAPI can be visu-
alized simultaneously. The third method
takes advantage of the newly described
chromosome painting probes. A target
chromosome can be readily identiFed
using a speciFc painting probe with a dif-
ferent fluorochrome that issues from the
gene to be mapped. Mapping of a new
gene to the chromosomal region from
which it was dissected is a crucial step
before any further molecular analysis is
done. The success of an
in situ
experiment
depends on several critical factors. The
factors that contribute signiFcantly to the
fluorescent signal include the speciFc ac-
tivity of the probe used, its length, and the
effective concentration of probe per slide.
±urthermore, conditions for chromosome
preparation, banding, and staining must
be optimized accordingly. Often, chro-
mosomes do not band well enough to
identify individual chromosomes. Over-
staining with the counterstaining dye may
quench or diminish the hybridization sig-
nal. Distortion of chromosomal bands
following the ±ISH procedure can be mini-
mized by performing GTG banding Frst.
±inally, the problem of background noise
can be considerably alleviated by selecting
narrow excitation band optical Flters.
5
Laser-capture Microdissection (LCM)
Molecular examination of pathologically
altered cells and tissues at the DNA,
RNA, and protein level has revolution-
ized research and diagnostics in pathology.
However, the inherent heterogeneity of
primary tissues with an admixture of var-
ious reactive cell populations can affect
the outcome and interpretation of molec-
ular studies. Microdissection of tissue
sections and cytological preparations is
being increasingly used for the isolation
of homogeneous, morphologically identi-
Fed cell populations, thus overcoming the
obstacle of tissue complexity.
In conjunction with sensitive analyti-
cal techniques, such as the polymerase
chain reaction, microdissection has aided
in the precise
in vivo
examination of cell
populations, such as carcinoma
in situ
or the malignant cells of Hodgkin’s dis-
ease, which are otherwise inaccessible for
conventional molecular studies. To fur-
ther implement this concept, the National
Cancer Institute developed laser-capture
microdissection. LCM is a technique that
permits the isolation of morphologically
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