44
Chromosome, Microdissection and Microcloning
micro-FISH
) is rapid and may help identify
complex karyotypes.
A new multicolor-banding technique has
been developed that allows the differ-
entiation of chromosome region–speciFc
areas at the band level. The technique is
based on the use of differently labeled
overlapping microdissection libraries. The
changing
fluorescence
intensity
ratios
along the chromosomes are used to as-
sign different pseudocolors to speciFc
chromosome regions. Weimer et al. have
used that advantage and have further re-
Fned micro-±ISH by combining the tech-
niques of fluorescence
in situ
hybridization
(±ISH) and chromosomal microdissection
in one experiment. Rearranged chromo-
somes were Frst identiFed by multicolor-
±ISH; the fluorophore-labeled derivative
chromosomes were directly isolated by
microdissection and reverse painted to
identify the breakpoints. Thus, identiF-
cation of the composition, origin, and
breakpoints of rearranged chromosomes
was rapidly accomplished.
6.2
Recombinant DNA Libraries Generated
from Microdissected Chromosome
Fragments
Recombinant DNA libraries can be gen-
erated from microdissected chromosomal
fragments either by direct microcloning
or by PCR-based methods as described
earlier.
Although
methods
in
current
use yield libraries that represent only
a small fraction of unique sequences
within the dissected region, these recom-
binant clones can be valuable as entry
sites into poorly deFned chromosomal re-
gions. Also, they may be used as probes to
identify and localize clones from genomic
libraries that contain large DNA inserts.
Chromosome microdissection has been
used increasingly in genomic projects to
‘‘Fll in’’ speciFc chromosome regions that
are poorly mapped. Microdissection of a
poorly deFned chromosomal region, fol-
lowed by cloning and sequencing unique
sequence clones, could generate a pool
of potential landmarks or tracts of DNA
sequences 200 to 250 bp in length. Such en-
tities, called
sequence-tagged sites
(STSs), are
potentially useful in establishing a physical
map(atthebasesequencelevel)ofhuman
chromosomes. Chromosome microdissec-
tion may be useful in constructing ‘‘contig
maps’’ (a series of clones representing
overlapping regions of the genome). Mi-
crodissected DNA is cloned and ampliFed
by PCR and used as a probe to screen
genomic libraries that contain larger DNA
inserts (e.g. cosmid, BAC or YAC libraries)
under conditions that suppress repeat-
sequence DNA.
6.3
Isolation of Transcribed Sequences Using
Microdissected DNA as a Probe
Mapping expressed genes to speciFc chro-
mosome regions can also be accomplished
using radioisotopically labeled microdis-
sected DNA as probes in hybridization
experiments with appropriate cDNA li-
braries. This calls for sequential screening
of cDNA libraries using plaque hybridiza-
tion methods. More recently, methods
such as the capture approach utilizing
biotin-labeled probes bound to strepta-
vidin with magnetic beads are useful. A
large number of inheritable diseases and
traits have been mapped to chromosome
regions using genetic linkage methods.
The isolation of causative genes can be
rapidly accomplished by using chromo-
some microdissection to generate a library
restricted to DNA sequences from the re-
gion associated with the disease. Once
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