"Frontmatter". In: Plant Genomics and Proteomics
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Christopher A. Cullis - Plant Genomics and Proteomics-J. Wiley & Sons (2004)
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 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 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 3 3 3 4 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 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 Download 1.13 Mb. Do'stlaringiz bilan baham: |
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