Chromosome, Microdissection and Microcloning
25
They are separated by flipping them apart
with a razor blade. The coverslip con-
taining the squash preparation is placed
in three successive ethanol washes (70%,
95%, absolute) for 1 min and either kept
in ethanol or air-dried; it may then be kept
in a moisture-free, airtight box until use.
Conditions of aging, Fxing, and storage
described for mammalian chromosomes
should be used when preparing
Drosophila
chromosomes.
3
Methods of Chromosome Microdissection
The methods used in microdissection
and isolation of chromosome fragments
are an extension of the methods and
instrumentation originally used for cell
microsurgery. Many of the cutting nee-
dles and nanoliter volume pipettes were
described nearly a half-century ago by De
±onbrune. De ±onbrune also described
the tools for fashioning micropipettes,
such as micromanipulators and the mi-
croforge. Edstr¨om and his colleagues later
reported the Frst deFnitive chromosome
microdissection experiment using chro-
mosome spreads in an oil chamber. The
oil chamber was placed on an upright mi-
croscope and microdissections and DNA
extractions were performed using curved
micropipette tips and nanoliter volumes.
Subsequently, microcloning experiments
were done in nanoliter aqueous drops un-
der oil. While this was an effective method,
particularly in skilled hands, Hagag and
Viola and others found the use of an in-
verted microscope and straight needles
to be technically easier and more gener-
ally applicable. Hagag and Viola described
and improved the method by utilizing
an inverted microscope equipped with a
high-magniFcation system. Other groups
have described microscopes equipped with
a laser microbeam for chromosome mi-
crodissection. The laser microbeam has
evolved into a new technology known as
laser-capture microdissection
(LCM). LCM
allows the collection of a minute amount
of DNA and RNA from a pure cell popu-
lation in a heterocomplex tissue. A brief
accoun
to
feacho
fthe
seme
thod
si
sde
-
scribed below.
3.1
Equipment
The equipment for chromosome microdis-
section includes a microscope, a pair of
micromanipulators, a micropipette puller,
a microinjector, micropipette holders, and
glass tubing to make microdissection nee-
dles and nanoliter transfer pipettes. A
microforge and a micropipette grinder
are optional for fashioning specialized mi-
cropipettes. An ideal microscope (upright
or inverted) should have the following fea-
tures: Frst, a high-intensity light source
with a short and efFcient light path; sec-
ond, a high numerical aperture phase
and fluorescence optics. The condenser
should permit a sufFcient working dis-
tance (10–20 mm) and should allow easy
interchange between the various optics
during manipulations. Third, several spec-
imen holders that permit easy access to
chromosomes on the slide are required.
These holders should be easily attachable
to the stage and capable of rotating freely to
align chromosomes with microdissection
pipettes. Other accessories (camera ports,
video ports, etc.) must not interfere with
accessibility to the micromanipulators and
micropipettes. At least one micromanip-
ulator is required for microdissection of
chromosomes. A second manipulator may
be handy for the simultaneous manipula-
tion of the chromosomal fragments after
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