26
Chromosome, Microdissection and Microcloning
dissection (e.g. fragment collection, trans-
fer to aqueous drops). Three-dimensional
mechanical or hydraulic micromanipula-
tors are best suited for this application.
A hydraulic
drive system
with
three-
axis motion and single-direction angu-
lar movement controls ensures smooth,
precise, vibration-free movement during
operation. Most micromanipulators are
equipped with a Fne drive joystick to re-
duce hand movement during dissection,
separation, and collection of fragments.
Microdissection
needles
or
micropi-
pettes
are
produced
from
borosilicate
capillary tubes (1 mm o.d., 0.65 mm i.d.)
using a two-stage micropipette puller (e.g.
Narshige PP83). A micropipette puller
helps prepare needles consistently with
a speciFcally programed tip diameter.
Dissecting needles and volumetric mi-
cropipettes are drawn to a long taper with
a tip diameter of approximately 0.5
µ
m.
The pipette ends must be flexible enough
to permit the tip of the needle to accept
pressure without breaking. This balance
between needle flexibility and sharpness
of the tip must be determined empir-
ically by the investigator. The diameter
of the micropipette can be estimated us-
ing a microscope and a microgrid (e.g.
Graticules Ltd., England). The dissecting
needle is Ftted into a microinjector (e.g.
Eppendorf) holder or a microsyringe. The
micropipettes are connected with Tygon
or Teflon tubing to the injector. The mi-
cropipette, tubing, and syringe may be
Flled with silicone fluid (which is inert for
most biochemical procedures), to provide
a closed injection–suction system. Chro-
mosome fragments are cut and pooled
into an aqueous drop of 1 to 2 mL. The
collection droplet is placed under a small
volume of silicone oil on a depression slide
or a moist chamber next to the dissection
slide.
3.2
Methods
3.2.1
Oil Chamber Method
Microdissection using the oil chamber
method was Frst described for dissect-
ing and microcloning of polytene chro-
mosomes and has been applied since
to the dissection of mouse and human
metaphase chromosomes. The oil cham-
ber consists of a one-piece glass slide that
measures 70
×
35
×
6mm
3
with a rect-
angular groove 25mm wide and 3mm
deep. Chromosome spreads are prepared
on 12
×
30
×
0
.
17 mm
3
coverslips. Glass
chambers and coverslips are washed in
hydrochloric acid and distilled water be-
fore use. The coverglass is placed on
the groove (chromosome spreads side
down). All microdissection and micro-
cloning operations are performed on the
lower surface of the coverslips using var-
ious dissection needles and volumetric
pipettes. The space between the coverslips
and the bottom of the chamber is Flled
with liquid parafFn. The liquid parafFn
used in the oil chamber also contains phe-
nol–chloroform droplets and buffers to
be used for extraction, solubilization, and
restriction enzyme digestions and subse-
quent microcloning. In this method, in-
vestigators used an upright phase contrast
microscope equipped with a long work-
ing distance condenser to view through
the thick oil chamber. ±ocusing was done
by moving the stage. Microdissection was
performed under a high-power dry objec-
tive (e.g. 40
×
). All other manipulations,
such as micropipetting, transfer of DNA
fragments to aqueous droplets, and DNA
extractions, were done with low-power ob-
jectives (10 and 20
×
). ±igure 1 represents
the oil chamber apparatus and micro-
cloning steps.
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