"Frontmatter". In: Plant Genomics and Proteomics

M. balbisiana “Pisang Klutuk Wulung,” have been constructed and made
available to the Consortium members as the standard cultivars for charac-
terization. Because banana is a monocot it is likely that the information from
the rice genome would be generally applicable to gene discovery in this
organism. Therefore, the expectation is that these BAC libraries will enable
the wealth of information from the rice genome sequence to be leveraged for
the ultimate benefit of smallholder farmers throughout the tropics.
cDNA C
LONING
The term cDNA is short for complementary DNA, because messenger RNA
(mRNA) cannot be cloned directly but a DNA copy of the mRNA can be
cloned. The conversion of mRNA to DNA is accomplished by the action of
reverse transcriptase and DNA polymerase (Gubler and Hoffman, 1983). 
The reverse transcriptase makes a single-stranded DNA complement of the
mRNA. The second DNA strand is generated by DNA polymerase. The
double-stranded product can be introduced into an appropriate plasmid or
lambda vector. The availability of high-throughput sequencing methods
opened the way to developing EST collections from cDNAs. ESTs are short
fragments of cDNAs that are usually biased toward the 3¢ end of the mRNA.
3 2
2. T
H E
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A S I C
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— A
C Q U I R I N G
F
U N C T I O N A L
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E N O M I C
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To generate ESTs it is not necessary to generate full-length sequences.
However, full-length cDNA clones can greatly facilitate both gene annota-
tion and functional studies and are an important resource. A scheme to
isolate full-length cDNA clones is given in Chapter 4.
The identification of the mRNA population in a given cell or tissue is
necessary to gain an understanding of gene expression in that cell or tissue.
Another important function is to compare mRNA populations when the
plant material has been subjected to a variety of perturbations, such as the
stress of pathogen invasion, to characterize the changes in gene expression
under these altered growth conditions. The analysis of differential gene
expression is covered in detail in Chapter 6. 
The mRNA can be fractionated before cloning to overcome the wide
range of messenger abundances within the mRNA population by the nor-
malization and subtraction of libraries. Alternatively, the mRNAs can be
characterized by using various lengths of “tags” that unambiguously iden-
tify a particular message and that also quantify the mRNA levels. These
methods include serial analysis of gene expression (SAGE) (Powell, 1998;
Madden et al., 2000) and massive parallel sample sequencing (MPSS
TM
)
(Brenner et al., 2000), which are discussed in greater detail in Chapter 6.
Alternatively, the mRNA differences can be visualized with differential
display methods.
S
UBTRACTION
L
IBRARIES
The individual mRNAs that are isolated from a tissue can be present in that
population over many orders of magnitude. To prevent the excess redun-
dant characterization of the same message, normalization or subtraction is
used to reduce the disparity of representation within these populations. The
construction of these modified libraries takes advantage of some combina-
tion of hybridization kinetics and amplification. For normalization, two
cDNA populations are hybridized to reduce the abundance of the most
prevalent messages. The remaining cDNAs are then cloned and either
sequenced or used in some other expression profiling experiment (Figure
2.4). Alternatively, suppressive subtracted hybridization (SSH) (Diatchenko
et al., 1998; Figure 2.5) involves the hybridization and amplification of the
cDNAs to physically remove the highly abundant sequences. This type of
reduction in the representation of the highly abundant messages in the
mRNA population is particularly necessary to identify rare messages. The
normalization of a library should result in all the sequences, whether origi-
nally abundant or rare, being present in about the same frequency after nor-
malization. SSH, on the other hand, will result in the selective representation
of sequences that are only in one of the cDNA libraries. The efficacy of these
methods must be weighed against the costs of redundant sequencing of large
numbers of ESTs. Because none of the subtraction/normalization methods
C
L O N I N G
S
Y S T E M S
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3 4
2. T
H E
B
A S I C
T
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C Q U I R I N G
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E N O M I C
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PCR with biotin
B
B
B
B
B
B
B
B
B
B
PCR inserts with biotin incorporation
cDNA library with F1 origin of replication
Denature inserts
Anneal together
Isolate single-stranded DNA
Block with Cotl, oligo dT and linkers
Capture on streptavidin
beads to remove
abundant single strand
cDNAs and biotin inserts
S
S
S
SB 
S
If abundant cDNAs
depleted then clones
sequenced
Check frequency of
abundant cDNAs in 
normalized library