"Frontmatter". In: Plant Genomics and Proteomics
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Christopher A. Cullis - Plant Genomics and Proteomics-J. Wiley & Sons (2004)
Partial cDNA CAP B B AAAAAAAA XTTTTTTTTTT RE site primer adapter Protected CAP RNase sensitive CAP - removed AAAAAAAA XTTTTTTTTT RE site primer adapter AAAAAAAAA XTTTTTTTTTT RE site primer adapter XTTTTTTTTTTT RE site primer adapter XTTTTTTTTTT RE site primer adapter GGGGGGGGGG GGGGGGGGGG Second primer adapter CCCCCCCCCC AAAAAAAA XTTTTTTTTTT RE site primer adapter CAP B B CAP B S Full length cDNA clones FIGURE 4.3. Schematic representation of a general strategy to select and clone full- length cDNAs: Biotinylation (B) of diol groups and synthesis of first-strand cDNA with a degenerate primer adapter including restriction enzyme (RE) site; RNase I digestion; full-length cDNA captured by streptavidin-coated magnetic beads (S); cDNA released from beads by RNA hydrolysis; first-strand cDNA tailed with oligo(dG); second-strand cDNA synthesis primed; cDNA cloned using the restriction sites introduced with the primers (Adapted from LabLink 2, No. 1 1998 http://www.cpg-biotech.com/lablink/lablink_2_1_1.html). annotation and for the identification of alternative splicing sites for RNAs. Three possible strategies for full-length sequencing are: ∑ Transposon mutagenesis: A transposon is randomly inserted, in vitro, into the cDNA insert, and primers designed from both sides of the transposon are used for sequencing. Sequencing a number of inde- pendent transposon sites will be sufficient to assemble the complete cDNA sequence. ∑ Concatenated cDNA sequencing (CCS): cDNA inserts are concate- nated into a BAC-size molecule (100 kb) and then sheared, cloned, and shotgun sequenced by standard procedures. The full-length cDNA sequences are then assembled and edited with the appropri- ate software. ∑ Primer walking: Primers are designed from 5¢ and 3¢ end sequences and used for a second round of sequencing. Additional primers are then made and used until the whole contiguous cDNA sequence is obtained. The availability of full-length cDNA clones opens new avenues for iden- tifying gene function and protein interactions. As detailed more fully in Chapter 6, the use of tagged full-length cDNAs in transgenic plants can be a first step in isolating and identifying the protein complexes that exist in vivo. Transgenics can also be used to develop a protein atlas of where in the cell each of the genes is expressed. A full-length cDNA can be tagged with a dye and the tagged probe transformed back into the plant under the control of its native promoter. The site of the fluorescence will indicate the organ or tissue where the gene is expressed, as well as the cellular localization of the protein. The reintroduction of the full-length cDNA into a plant can also result in either overexpression or silencing of that gene. The subsequent phenotype that is observed provides clues as to the function of the gene. In addition, overexpression of such a gene, for which a full-length cDNA is available, can be accomplished in a heterologous system, such as yeast or E. coli, followed by in vitro studies of the protein function. Thus, although ESTs are useful for the initial description of the tran- scriptome, the full-length cDNAs are a much more valuable resource. The availability of full-length cDNAs for many species will greatly enhance the rate of gene discovery, patterns of splicing, and the understanding of gene function in plants. Download 1.13 Mb. Do'stlaringiz bilan baham: |
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