Chromosome, Microdissection and Microcloning
31
to the proteinase K drop using another vol-
umetric pipette. The aqueous droplets will
be used later for washing and solubilizing
extracted DNA. For easy manipulation, all
these steps should be performed at low
magni±cation (10
×
). Coverslips contain-
ing the metaphase spreads are secured
into the customized slide holder, spreads
side up, and mounted onto the rotating
stage. Usable spread(s) are identi±ed and
marked at 10
×
magni±cation for easy ori-
entation of the chromosome of interest.
The dissecting needle is placed within a
few micrometers of the chromosome of
interest in the metaphase spread and at
10
×
magni±cation. The system is switched
to high magni±cation (40–60
×
)andvideo
display (3200–5000
×
). After the desired
chromosome has been identi±ed, the GTG
band to be dissected is approached at an
angle of about 30 to 45
and the dissec-
tion needle is brought up into the ±eld
of the spread. Dissections are preferably
performed in cells with well-spread chro-
mosomes and when the chromosome of
interest is located in the periphery of
the metaphase spread. This precaution
minimizes cross-contamination with other
chromosomal material.
Microdissection is performed with the
aid of the micromanipulator by moving
the micropipette tip back and forth across
the chromosome band of interest. Under
these conditions, the chromosomes are
readily cut across, and the dissected region
is detached from the slide. Each dissec-
tion leaves a clear zone in the surrounding
cytoplasm, thus allowing easy and reliable
veri±cation of the dissected chromosomes.
Fragments that are not identi±able are dis-
carded. The selected band is scraped off
the slide, and the fragment is pushed to
the side with a micropipette. A drop of oil
is expelled from the micropipette to cover
the fragment and provide a closed system
during suction and collection of the frag-
ment. The fragment is secured to the tip of
the micropipette by applying suction and
a holding pressure that is maintained dur-
ing fragment transfer. The microscope is
then switched to low magni±cation and the
depression slide (containing DNA collec-
tion and extraction drops) is brought into
the ±eld of view. The micropipette is gen-
tly introduced into the oil drop containing
the aqueous droplets, while the holding
pressure is maintained. The fragment is
expelled into the droplet containing pro-
teinase K/SDS solution by reversing the
micrometer into the injection mode. The
fragment should be visible in the drop at
this point. This process is repeated un-
til a suf±cient number of fragments have
been collected. A schematic representa-
tion of the sequence of these steps appears
in Fig. 4. An example of microdissection
of human chromosome 3 at bands 3p13,
3p14, 3p21, and 3q21 using the videomi-
croscopy method is illustrated in Fig. 5.
Thet
imerequ
iredtom
icrod
issec
taspe
-
ci±c chromosome region depends on the
quality and number of metaphase spreads
on the coverslip. With some experience,
10 to 15 fragments can be dissected per
hour. Approximately 30 to 100 fragments
are suf±cient for cloning experiments.
4
Methods of Cloning and Analysis of
Microdissected DNA
4.1
Cloning Methods
The ±rst experiments to successfully gen-
erate recombinant genomic DNA clones
directly from microdissected eukaryotic
chromosome fragments were performed
in the early 1980s by Jan-Erik Edstr¨om and
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