"Frontmatter". In: Plant Genomics and Proteomics

Partial repair and
transform
normalized
library
FIGURE 2.4.
Normalization of cDNA libraries. The cDNA library is divided into
two sets, one in which the inserts are amplified with the incorporation of biotin, and
the other being the isolation of single-stranded circles. The two are appropriately
annealed together, and the resulting mixture is bound to streptavidin beads. 
The unbound circles (low-abundance cDNAs in the library) are repaired and 
retransformed. The normalized library is then sequenced. (Adapted from
http://hg.wustl.edu/COGENE/INFO/norm.html).
FIGURE 2.5.
SSH scheme (from http://www.clontech.com/products/literature/
pdf/brochures/PCR-SelectBR.pdf). The 2 samples to be subtracted are isolated and
converted to cDNA. The tester sample is divided into 2, and each fraction ligated to
a specific adaptor. The tester is then hybridized with excess driver cDNA that did
not have adaptor added. After the first hybridization the samples are mixed, 
additional driver is added, and the mixture is hybridized again. The ends of the 
molecules are then filled in and amplified by using the adaptor sequences as primers.
Only those messages that are rare in the tester sample are amplified. These can 
then be cloned and sequenced. (Reprinted with permission from BD Biosciences
Clontech.)


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C
L O N I N G
S
Y S T E M S
3 5
RNA sample 1
(Tester)
RNA sample 2
(Driver)
cDNA synthesis
Digest with RsaI
Ligate tester with 2
different adaptors
Tester cDNA with Adaptor 1
Tester cDNA with Adaptor 2
Excess driver cDNA
First hybridization
a
b
c
d
a, b, c, d 
+
e
Second hybridization: samples
mixed additional driver added
Fill in the ends
a
b
c
d
e
Add primers
Amplify
a, d - no amplification
b - no amplification
c -linear amplification
e -exponential amplification
and

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completely remove the abundant messages, there will still be a significant
waste in sequencing the subtracted libraries as the common messages will
already have been identified. 
D
IFFERENTIAL
D
ISPLAY
Differential display technology works by the amplification of the 3¢ terminal
portions of mRNAs and the visualization of those fragments on a DNA
sequencing gel (Liang and Pardee, 1992). The anchored oligo-dT primers
define the 3¢ end of the RNA, and then a limited number of short arbitrary
primers are used to amplify most of the mRNA in a cell (Figure 2.6). The
separation of the amplified fragments by denaturing polyacrylamide 
gel electrophoresis allows direct side-by-side comparison of most of 
3 6
2. T
H E
B
A S I C
T
O O L B O X
— A
C Q U I R I N G
F
U N C T I O N A L
G
E N O M I C
D
ATA
RNA sample 1
RNA sample 2
Single strand cDNA template
First strand
cDNA synthesis
Amplification using pairwise combinations of short primers
3 cycles of low stringency PCR
25 cycles of high stringency PCR
Label included for vizualization of PCR products
Electrophorese on acrylamide gel
Expose gel to x-ray film
Primer pairs 
RNA sample 1 2
1 2 1 2
1 2 1 2
1 2
a
b
c
d
e
f
FIGURE 2.6.
Differential display. Each sample is treated in the same way. The first-
strand cDNA is synthesized. This is then amplified with a series of short primers 
for 3 cycles at low stringency to account for the length of the primers. After the 
first 3 cycles the amplification is continued for a further 25 cycles at high stringency.
In the amplification, label is included so that the products can be visualized. The
products are then separated on a polyacrylamide gel, and the bands are compared.
Differential bands can be excised and characterized further.

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the mRNAs. The polymorphic bands representing the differentially
expressed sequences can be excised from the gel, reamplified, cloned, and
sequenced.
DNA M
ICROARRAYS
The combination of sequence information and automation has facilitated the
change from looking at one gene at a time to being able to profile expression
and changes of expression for many genes. DNA microarrays, which are one
of the basic formats for looking at these changes, are orderly arrangements
of DNA samples fabricated by high-speed robotics on glass, nylon, or other
substrates. The initial experiments used cDNA immobilized on the surface
with robot spotting, although a current alternative to the use of PCR prod-
ucts is oligonucleotides either synthesized on the chip or synthesized off the
chip and anchored later. These arrays can be used to detect polymorphisms
and mutations, as well as to help map genomic libraries and characterize
gene expression. 
The schema for a microarray experiment is shown in Figure 6.2. The
steps involved are:
1. Development of the microaray. As mentioned above, this can be an
array based on either fragments amplified through PCR or small
oligonucleotides that are attached to the substrate. In either case, the
sequences to be placed on the microarray must be selected. The PCR
products, for example, can be amplified from cDNAs that comprise a
unigene set developed from EST collections (see Chapter 4 and Figure
4.1) or from genomic DNA by using primers based on gene predic-
tions from a genomic sequence. The oligonucleotides can be designed
from the same information source. 
2. The arrays are then printed by using the information. When the PCR
products or the oligonucleotides are first generated and then attached
to a substrate, this results in a more flexible platform because the
array designed can be changed relatively easily and less expensively
than when the oligonucleotides are actually synthesized on the 
substrate. 
3. The probes are labeled and hybridized to the microarrays. The design
of the experiment and the number of replicates are important para-
meters for any differences observed to be statistically validated. 
4. The hybridization data are analyzed with a series of software pro-
grams, and any patterns of coordinated changes in gene expression
are detected. 
5. The form in which the data are reported is important for comparison
between experiments. A series of standards for such reporting have
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3 7

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been developed, and are continuing to be developed, and are known
as the minimal information about a microarray experiment (MIAME;
Brazma et al., 2001).