"Frontmatter". In: Plant Genomics and Proteomics
Download 1.13 Mb. Pdf ko'rish
|
Christopher A. Cullis - Plant Genomics and Proteomics-J. Wiley & Sons (2004)
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. Download 1.13 Mb. Do'stlaringiz bilan baham: |
Ma'lumotlar bazasi mualliflik huquqi bilan himoyalangan ©fayllar.org 2024
ma'muriyatiga murojaat qiling
ma'muriyatiga murojaat qiling