48
Chromosome, Microdissection and Microcloning
gene-transfer experiment in 1989. Human
centromeric DNA was successfully trans-
f
e
r
r
edt
of
e
r
t
i
l
i
z
edm
ou
s
eo
v
aandw
a
s
detected in blastocysts, 15-day embryos,
and a mouse was derived from an in-
jected ovum. Microinjection of insoluble
or partially solubilized chromatin particles
is likely to be an inefFcient vehicle for
gene transfer. A microscope stage such as
that designed by Hagag and Viola would
overcome this problem. The circular ro-
tating stage accommodates a depression
slide to collect chromosome fragments
and a culture dish to contain fertilized
ova (±ig. 2b). The stage permits the col-
lection of a large number of fragments.
The DNA is solubilized using trypsin or
proteinase K in a small aqueous drop that
is placed under oil. Soluble DNA is then
used in microinjection of ova during the
same setting.
6.7
Coupling Microdissection with Microarrays
and Proteomics Technologies
(DNA/Protein Microchips)
The microarray expression analysis system
has become one of the most widely used
functional genomic tools. The system has
features that make it widely used for
proFling mRNA expression. Microarrays
are constructed by mechanically arraying
PCR-ampliFed cDNA clones or genes
representing the collection of genes to be
assayed at a high density on derivatized
glass microscope slides. A relatively simple
x
-
y
-
z
stage robotic system is used to
spot
an
entire
set
of
genes
from
a
microbial genome or tens of thousands of
eukaryotic cDNA clones. The microarrays
are queried in cohybridization assays using
two or more fluorescently labeled probes
prepared
from
the
cellular
phenotype
of interest.
Hybridization is assayed using a confo-
cal laser scanner to measure fluorescence
intensities, which allows simultaneous de-
termination of the relative expression lev-
els of all the genes represented in the array.
Expressed sequence tags (EST) are single-
pass, partial sequences of cDNA clones
used for gene discovery, identiFcation,
and mapping in humans and other organ-
isms. Generally, cDNA clones are selected
to represent as many unique transcripts
as possible. The two most widely used
ESTs are UniGene at the National Cen-
ter for Biotechnology Information (NCBI)
and
the
Institute
for
Genomic
Re-
search
(TIGR)
Human
Gene
Index
Readers
new
to
this
technology
may
consult the excellent concise guide by
Hegde et al. The article offers a discus-
sion of protocols, instrumentation, and
data analysis. Several microarray systems
are commercially available in the mar-
ket. The ScanArray 3000 (GSI Lumonics,
Watertown, MA, USA) and Biorad Ver-
sArray (www.bio-rad.com/versarray) are
examples. The affymetrix (Santa Clara, Cal-
ifornia) DNA chip is widely used.
A recent study has put to use the
combination of microarrays and microdis-
section to elucidate the distinct molecular
changes that occur at each progressive
stage of prostate cancer. The cDNA gen-
erated from prostate cancer (tumor) and
benign tissue (reference) samples were
labeled with distinguishable fluorescent
dyes and hybridized to a cDNA microarray
consisting of known sequence motifs, each
representing a gene of interest. The cDNA
microarray was analyzed using a scanner,
and fluorescence ratios were determined
for each gene. Ratios were then imported
into a database and analyzed using statisti-
cal algorithms. The data surveying method
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