Chromosome, Microdissection and Microcloning
39
a 99% probability of cloning the entire
10 Mb fragment is determined as follows:
N
=
ln
(
1
0
.
99
)
ln[1
(
2
.
4
10
3
/
10
7
)
]
=
19 000
The actual number of clones obtained
would be determined by factors such as
the efFciency of the cloning method, the
cloning capacity of the vector, the relative
number and size of clonable and nonclon-
able DNA sequences in the target DNA,
and the degree of DNA degradation during
the process of chromosome preparation
and library construction. Therefore, the
percentage of the total DNA that has been
cloned would be determined as follows:
% fragment DNA cloned
=
experimental number of clones
obtained – mean average
insert size
fragment DNA content
×
100
Evolutionarily conserved DNA sequences
are indicative of potential structural gene
sequences. To determine the
potential
structural genes
in the library, the gene(s)
of interest can be hybridized to genomic
DNA from a number of species using rel-
atively stringent hybridization conditions.
The relatedness of a sequence among var-
ious species is readily identiFed by its
hybridization to genomic DNA from these
species. Genomic DNA from a number
of species is now commercially available
on biomembrane blots (e.g. Biomap Evo
blots, BIOS), sometimes also referred to
as
Zooblots
. These blots contain genomic
DNAf
romhuman
,ca
t
,dog
,sheep
,p
ig
,
cow, rabbit, rat, and mouse. Potential
structural genes are identiFed as those that
hybridize to DNA sequences from at least
three species. Once a structural gene has
been recognized, its sequence can be used
to isolate the full-length expressed gene
from a cDNA library of the appropriate
tissue. Alternatively, structural genes may
be identiFed by directly hybridizing cloned
genomic DNA to cDNA libraries under
conditions that suppress hybridization of
repeat sequences. ±ollowing the detailed
analysis of the recombinant clones, it is
also necessary to perform
in situ
hybridiza-
tion to conFrm whether the clones map to
the microdissected chromosomal region of
interest. In addition to identifying the chro-
mosome on which a gene lies, fluorescence
in situ
hybridization (±ISH) helps position
the gene of interest at a resolution of about
1t
o2
×
10
7
bp (0.5%) of the genome.
Genomic clones as well cDNA clones can
be used in ±ISH. Unique sequences con-
tained in large-insert DNA probes, such as
cosmid and YAC clones, can be used di-
rectly in ±ISH following the suppression
hybridization of repetitive sequences Frst
with unlabeled genomic DNA or repeat
sequences (e.g.
Cot
1DNA)
.
The fluorescent labeling system is based
on the modiFcation of the DNA probe with
biotin. The probe DNA is labeled using
nick translation. Biotin is detected by its
binding to avidin or streptavidin coupled to
enzymes such as horseradish peroxidase
or to fluorochromes. ±luorescent
in situ
hybridization of metaphase chromosome
spreads involves two steps:
1. Denaturing the two strands of the
chromosomal
DNA
contained
in
a
metaphase spread on a coverslip, fol-
lowed by hybridization to a denatu-
red fluorescent-labeled oligonucleotide
probe.
2. Localization of the hybridization signal
to a speciFc chromosomal band region.
SigniFcant ampliFcation of the signal can
be achieved using a second layer of a bio-
tinylated antiavidin antibody and a third
previous page 1359 Encyclopedia of Molecular Cell Biology and Molecular Medicine read online next page 1361 Encyclopedia of Molecular Cell Biology and Molecular Medicine read online Home Toggle text on/off