Chromosome, Microdissection and Microcloning
43
expression profling, and single-cell ap-
plications. With regard to the technology
itselF, protocols, sample preparation, and
signing up For training, visiting the Fol-
lowing NIH website is recommended.
6
Applications of Chromosome and Tissue
Microdissection
A recent three-year period literature review
oF the potential uses oF DNA (RNA and pro-
tein) obtained by microdissection resulted
in more than 600 reports on the application
oF chromosome and tissue microdissec-
tion to generate recombinant DNA clones
From various human, mouse, rat, plant,
and a number oF other species. With the
inclusion oF LCM, there is a plethora oF
articles on the use oF RNAs and proteins
derived predominantly From human tissue
archives and biopsies to delineate several
diseases. The applications oF microdissec-
tion can be grouped into eight categories.
1. Direct analysis oF microdissected chro-
mosomal DNA Following PCR amplif-
cation.
2. Generation oF recombinant DNA li-
braries From specifc chromosomal re-
gions.
3. Isolation
oF
expressed
genes
local-
ized
to
specifc
regions by
probing
cDNA
libraries
with
amplifed
mi-
crodissected DNA From that region.
4. Study oF chromosome abnormalities in
cancer cells
5. Investigation oF other specialized chro-
mosomal structures
6. Gene
transFer
using
chromosome
Fragments
7. Coupling
microdissection
with
mi-
croarrays and proteomics technologies
(DNA/protein microchips)
8. Incorporation oF microdissection tech-
niques in modern pathology
6.1
Direct Analysis of the PCR Product of
Microdissected Chromosome Fragments
IF the sequence oF the gene oF inter-
est is known, gene-specifc oligodeoxynu-
cleotide primers can be synthesized and
used in a PCR diagnostic For that gene.
DNA From a microdissected Fragment
serves as a PCR template using gene-
specifc primers to localize (or exclude)
a gene From that chromosome region.
Also, iF the spatial relationship oF multi-
ple genes in a specifc chromosome region
is known, target sequences can be ampli-
fed by PCR to detect structural alterations
(deletions or rearrangements). In these
experiments, a limited number oF chro-
mosome Fragments From one region are
dissected and target sequences are am-
plifed using site-specifc oligonucleotide
primers. The inability to generate a PCR
product using DNA From the aFFected
region (in the presence oF appropriate posi-
tive controls) is indicative oF the absence or
rearrangement oF the gene in that region.
Using this method, it was possible to map
the breakpoints on chromosome 11 that
are present in a number oF translocations
associated with human leukemia. When
a translocation is identifed by karyotype
analysis but the chromosome oF origin oF
the derivative chromosome is ambiguous,
analysis oF the microdissected transloca-
tion site may be helpFul. One strategy
involves the microdissection oF the region
oF translocation, Followed by
in vitro
ampli-
fcation (PCR) using nonspecifc primers.
The PCR product is fluorescently labeled
and hybridized to a normal karyotype to
identiFy the chromosome that participated
in the translocation. This method (called
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