"Frontmatter". In: Plant Genomics and Proteomics

T
HE
G
ENERATION OF
F
ULL
-L
ENGTH
cDNA
S
Full-length cDNAs are an essential resource for the functional analysis of
plant genes (Figure 4.2). The confirmation of the prediction of transcription
units from genomic sequence data is dependent on validation with a full-
length cDNA. Full-length cDNAs are also necessary for the correct identifi-
cation of splice sites. The occurrence of alternate splicing events also must
be confirmed by the identification of a full-length cDNA containing the alter-
natively spliced region. The full-length cDNA can be used in both homolo-
gous and heterologous expression systems to generate large amounts of
protein for functional and structural studies to determine the function of the
gene. In addition, sequencing of the full-length transcripts will allow the
identification of RNAs from different members of gene families.
Full-length cDNA library construction is more technically challenging
compared with EST generation. A full-length first-strand cDNA is not 
efficiently produced by reverse transcription, especially if the mRNA has 
a stable secondary structure. Libraries made from cDNAs, therefore, can
T
H E
G
E N E R AT I O N O F
F
U L L
- L
E N G T H C
D N A
S
7 3

---

7 4
4. G
E N E
D
I S C O V E R Y
TABLE 4.1. EST 
NUMBERS IN DB
EST 
AS OF
2/14/2003 
FOR SELECTED
PLANT SPECIES
Wheat
415,589
Hordeum vulgare + subsp. vulgare (barley)
314,882
Glycine max (soybean)
308,564
Zea mays (maize)
197,921
Medicago truncatula (barrel medic)
180,939
Arabidopsis thaliana (thale cress)
178,464
Lycopersicon esculentum (tomato)
148,554
Oryza sativa (rice)
130,772
Solanum tuberosum (potato)
94,423
Sorghum bicolor (sorghum)
84,712
Lactuca sativa (lettuce)
68,188
Pinus taeda (loblolly pine) 
60,226
Populus tremula ¥ Populus tremuloides
56,013
Helianthus annuus (sunflower)
46,951
Gossypium arboreum 
38,894
Lotus japonicus 
33,124
Vitis vinifera (grape)
30,940
Ipomoea nil (morning glory)
25,899
Mesembryanthemum crystallinum (common ice plant) 
25,446
Hordeum vulgare subsp. spontaneum
24,150
Populus balsamifera subsp. trichocarpa 
23,717
Capsicum annuum (pepper)
22,433
Sorghum propinquum 
21,387
Phaseolus coccineus 
20,120
Beta vulgaris (beet) 19,617
Populus tremula
14,078
Gossypium hirsutum (upland cotton)
10,716
Prunus persica
10,185
Zinnia elegans 
9,783
Triticum monococcum
9,572
Secale cereale (rye)
8,930
Lycopersicon pennellii
8,346
Oryza minuta
5,268
Brassica rapa subsp. pekinensis (Chinese cabbage)
4,316
Rosa hybrid cultivar 
2,874
Prunus dulcis
2,858
Brassica napus (oilseed rape)
2,691
Citrus sinensis 
2,623
Lycopersicon hirsutum
2,504
Mentha x piperita (peppermint)
1,316
Linum usitatissimum (flax)
1,299
Allium cepa (onion)
1,193
Cicer arietinum (chickpea)
23
Narcissus pseudonarcissus (daffodil)
1

---

contain both full-length and partial cDNAs. Efficient techniques exist for
selecting only full-length cDNAs. One such method for constructing cDNA
libraries with a high content of full-length clones involves starting from the
first transcribed nucleotide (Figure 4.3). A biotin label for the cap structure,
based on the principle that the cap site and 3¢ end of mRNA are the only
sites that carry the diol structure, has been developed. The diol groups 
at each end of the mRNA are biotinylated, and then the first-strand cDNA
is synthesized. This synthesis is primed with a degenerate primer
[XTTTTTTTT(Restriction site)]. The reaction mixture is then digested with
RNase, and only the full-length cDNAs are protected from degradation of
the unpaired mRNA. Therefore, the 5¢ ends of all the partial cDNAs are
removed (along with the biotin) as are the 3¢ ends of all molecules. The full-
length cDNAs are captured on streptavidin-coated magnetic beads, and the
cDNA is released from the beads and the mRNA by treatment with RNase
H and alkaline hydrolysis. The cDNA is then tailed with oligo(dG) that is
used to prime the second-strand synthesis. Again, this primer also has an
extension that includes a restriction enzyme site. After the second-strand
synthesis the full-length cDNA is cloned with the restriction sites inserted
with the first- and second-strand primers.