Biomedical Engineering Reference
In-Depth Information
Box 6.5 Differential screening with DNA chips
and most genes of the yeast
Saccharomyces
cerevisiae
(De Risi
et al
. 1997). This has allowed
comprehensive parallel analysis of the expression of
all genes simultaneously in a variety of experimental
assays (Cho
et al
. 1998, Chu
et al
. 1998, Spellman
et al
. 1998, Jelinsky & Samson 1999). It is likely that
complete genome arrays will be available for higher
eukaryotes, including humans, within the next few
years, offering an unprecedented ability to capture
functional snapshots of the genome in action.
cDNA microarrays
Miniaturization and automation have facilitated
the development of DNA microarrays, in which
DNA sequences are displayed on the surface of a
small 'chip' of either nylon or glass. In the initial
description of this technology (Schena
et al
. 1995),
up to 10 000 cDNA clones, each in the region of
several hundred nucleotides in length, could be
arrayed on a single microscope slide. The cDNAs
were either obtained from an existing library or
generated
de novo
by PCR. In each case, the machine
transfers a small amount of liquid from a standard
96-well microtitre plate on to a poly-l-lysine-coated
microscope slide, and the DNA is fixed in position
by UV irradiation. Arrays are used predominantly for
the multiplex analysis of gene expression profiles.
Total RNA is used to prepare fluorescently labelled
cDNA probes and signals are detected using a laser.
Each hybridization experiment generates a large
amount of data. Comparisons of expression profiles
generated using probes from different sources can
identify genes that are differentially expressed in
various cell types, at different developmental stages
or in response to induction (reviewed by Schena
et al
. 1998, Xiang & Chen 2000). There have been
many successes with this relatively new technology,
including the identification of genes involved in the
development of the nervous system (Wen
et al
. 1998)
and genes involved in inflammatory disease (Heller
et al
. 1997). Arrays have been constructed including
every gene in the genome of
E. coli
(Tao
et al
. 1999)
Oligonucleotide chips
An alternative to spotting presynthesized cDNAs or
ESTs on to slides is to synthesize oligonucleotides
in situ
on silicon or glass wafers, using similar
processes to those used in the manufacture of
semiconductors (Lockhart
et al
. 1996, Shalon
et al
.
1996). Using current techniques, up to 1 000 000
oligonucleotides can by synthesized in tightly
packed regular arrays on chips approximately
1cm
2
(Lipshutz
et al
. 1999). Unlike cDNA arrays, a
hybridizing probe sequence is recognized not by a
single cognate cDNA, but by a combination of short
oligonucleotides, from which its sequence can be
deduced (reviewed by Southern 1996a,b). Chips
are more versatile than arrays, because they can be
used not only for expression analysis but also for
DNA sequencing (resequencing) (Chee
et al
. 1996)
and the analysis of differences between genomes
at the level of single nucleotide polymorphisms
(Hacia 1999).
described which use pairs of short arbitrary primers
to amplify pools of partial cDNA sequences. If the
same primer combinations are used to amplify
cDNAs from two different tissues, the products can
be fractionated side by side on a sequencing gel, and
differences in the pattern of bands generated, the
mRNA fingerprint
, therefore reveal differentially
expressed genes (Fig. 6.17). Essentially, the distinc-
tion between the two techniques concerns the
Difference cloning by PCR
Displaying differences - differential-display
PCR and arbitrarily primed PCR
As expected, PCR-based methods for difference
cloning are more sensitive and rapid than library-
based methods, and can be applied to small amounts
of starting material. Two similar methods have been
