24
Chromosome, Microdissection and Microcloning
acid fxation For chromosome microdis-
section studies has one major adverse
eFFect: it causes depurination and nick-
ing oF DNA. Depurinated DNA is more
susceptible to enzymatic digestion as well
as to the DNA degradation associated with
chromosome aging. ThereFore, a number
oF precautions and modifcations oF stan-
dard fxation protocols should be observed.
Since acid depurination is a hydration re-
action, acetic acid and methanol should be
Free oF water. Reagents should be aliquoted
into small bottles and kept well sealed. All
steps in chromosome fxation and spread-
ingshouldbedoneat0to4
C. Care should
be taken to avoid prolonged exposure oF
chromosomes to fxative. Depending on
the cell type, cells may be kept in fxative
For a Few seconds to 10 min beFore spreads
are dropped onto coverslips.
2.1.4
Aging
Once metaphase spreads have been fxed
and dropped onto coverslips, the chromo-
somes must be aged to obtain optimum
GTG banding. Aging is accomplished by
keeping chromosome spreads at room
temperature For 3 to 5 days or at 56
C
overnight. The precise physicochemical
basis For the aging oF chromosomes is
unknown, but it is evident that chromo-
somal DNA that has become depurinated
by acid fxation grows increasingly sin-
gle stranded and is degraded during the
aging process. ThereFore, when possi-
ble, it is preFerable to microdissect Fresh,
uniFormly stained (solid staining) or un-
stained chromosomes. This procedure is
certainly applicable to the microdissection
oF telomeric or centromeric regions oF
chromosomes, which are readily recogniz-
able by size and morphology. Similarly, the
microdissection oF marker chromosomes
and double minutes can be perFormed with
unstained material.
2.1.5
GTG Banding for Chromosome
Microdissection
GTG banding oF metaphase chromosomes
is the most commonly used technique
when chromosomes must be stained For
identifcation oF a specifc chromosome
or chromosome region. Coverslips are re-
moved From ethanol and are placed in a
sterile Coplin jar containing pH 6.8 buFFer
at room temperature. The coverslips are
immediately removed From the buFFer and
are placed in sterile 0.5% trypsin/EDTA
solution (in Hanks buFFered salt solution),
and incubated at room temperature For
1 to 3 min. IF solid staining is desired,
the trypsin/EDTA step is omitted. The
coverslips are washed For 1 s in pH 6.8
buFFerandthenplaceddirectlyintoGiemsa
stain. Staining may take 15 to 90 s. The
coverslips are washed twice in pH 6.8
buFFer and air-dried. The optimum time
For trypsin treatment and staining should
be determined experimentally. AFter a cov-
erslip has been air-dried, DNA will begin
aging. ThereFore, to obtain high-quality
DNA, it is recommended that microdis-
section be perFormed immediately aFter
staining. Also, to avoid contaminating
slides with Foreign DNA, the staining pro-
cedure should be perFormed in a biological
containment hood. Sumner has reviewed
chromosome banding.
2.2
Preparation of Dipteran Chromosomes
Drosophila
chromosomes may be prepared
From dipteran salivary gland For microdis-
section. Salivary glands are dissected in
insect Ringer’s solution and are spread
on a siliconized coverslip in a drop oF
45% acetic acid. The tissue is squashed
between two siliconized coverslips. The
covers
l
ipsarethenhe
ldinp
lacew
i
thFor
-
ceps and quick-Frozen in liquid nitrogen.
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