22
Chromosome, Microdissection and Microcloning
unit of the general formula D (T/A
1
4
dG
1
8
)
.
The
terminal
repeat
number
varies considerably in different species,
from a few hundred base pairs in ciliates to
10 kb in humans, and up to 150 kb in mice.
This review of chromosome structure was
conFned to essential background, and
more detailed discussion of this subject
can be found elsewhere.
2
Preparation of Chromosomes for
Microdissection
Methods of preparing chromosomes and
chromosome bands have been empiri-
cally developed by cytogeneticists over
the last four decades. These methods,
however, must be modiFed when chro-
mosomes are to undergo microdissection.
Conventional banding and staining tech-
niques result in degradation of chromo-
somal DNA and hence render the DNA
unsuitable for biochemical and biolog-
ical studies. In fact, it is evident that
improvement in GTG banding is asso-
ciated with increased DNA degradation.
A number of factors contribute to DNA
breakdown during the preparation of chro-
mosome spreads. DNA appears to be
more unstable (i.e. susceptible to denatu-
ration and DNAse digestion) in condensed
metaphase chromatin than it is in inter-
phase chromatin. ±urthermore, at each
step in the preparation of metaphase chro-
mosomes – that is, hypotonic swelling,
Fxing, and ‘‘aging’’ – DNA degradation is
facilitated.
Chromosome Fxation and aging con-
tribute most signiFcantly to chromosomal
DNA degradation. Therefore, it is essential
in microdissection experiments to con-
sider chromosomes as units of linear DNA
rather than cytogenetic structures.
Several critical aspects of chromosome
preparation for microdissection experi-
ments are worth mentioning. These in-
clude the enrichment of preparations for
metaphase spreads, hypotonic treatment,
chromosome Fxation, aging, staining, and
storage of chromosomal DNA. When plan-
ning for microdissection, it is ideal to
have as many metaphase spreads as pos-
sible on one coverslip. Only a fraction of
the spreads are amenable to dissection
because of the poor extension of chro-
mosomes in some spreads, overlapping
chromosomes, or disadvantageous orien-
tation of the chromosome of interest. The
number of metaphase spreads may be in-
creased by synchronization in the cell cycle
to capture the maximum number of cells
in mitosis. In most instances, adequate
numbers of mitotic cells can be obtained
from short- or long-term monolayer cul-
tures without drug synchronization by
plating cells sparsely and harvesting mi-
totic cells when cells are in a log phase
of growth. If necessary, the thymidine
‘‘block’’ and ‘‘release’’ method may be
used. The use of synchronizing agents
such as antimetabolites (e.g. methotrex-
ate), cytotoxic drugs (e.g. actinomycin D),
or other chemicals capable of inducing
DNA damage is not advised.
2.1
Aspects of Preparing Mammalian
Chromosomes
2.1.1
Sources of DNA
Several
sources
can
be
used
to
pre-
pare chromosomal DNA for microdissec-
tion. If whole-blood microculture is used,
metaphase spreads are prepared from pe-
ripheral blood T lymphocytes as follows.
Venous blood is drawn into a heparinized
tube or into a syringe containing 0.1 mL of
heparin stock solution (5000 IU mL
1
)
.
previous page 1342 Encyclopedia of Molecular Cell Biology and Molecular Medicine read online next page 1344 Encyclopedia of Molecular Cell Biology and Molecular Medicine read online Home Toggle text on/off