"Frontmatter". In: Plant Genomics and Proteomics
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Christopher A. Cullis - Plant Genomics and Proteomics-J. Wiley & Sons (2004)
Partial repair and transform normalized library FIGURE 2.4. Normalization of cDNA libraries. The cDNA library is divided into two sets, one in which the inserts are amplified with the incorporation of biotin, and the other being the isolation of single-stranded circles. The two are appropriately annealed together, and the resulting mixture is bound to streptavidin beads. The unbound circles (low-abundance cDNAs in the library) are repaired and retransformed. The normalized library is then sequenced. (Adapted from http://hg.wustl.edu/COGENE/INFO/norm.html). FIGURE 2.5. SSH scheme (from http://www.clontech.com/products/literature/ pdf/brochures/PCR-SelectBR.pdf). The 2 samples to be subtracted are isolated and converted to cDNA. The tester sample is divided into 2, and each fraction ligated to a specific adaptor. The tester is then hybridized with excess driver cDNA that did not have adaptor added. After the first hybridization the samples are mixed, additional driver is added, and the mixture is hybridized again. The ends of the molecules are then filled in and amplified by using the adaptor sequences as primers. Only those messages that are rare in the tester sample are amplified. These can then be cloned and sequenced. (Reprinted with permission from BD Biosciences Clontech.) C L O N I N G S Y S T E M S 3 5 RNA sample 1 (Tester) RNA sample 2 (Driver) cDNA synthesis Digest with RsaI Ligate tester with 2 different adaptors Tester cDNA with Adaptor 1 Tester cDNA with Adaptor 2 Excess driver cDNA First hybridization a b c d a, b, c, d + e Second hybridization: samples mixed additional driver added Fill in the ends a b c d e Add primers Amplify a, d - no amplification b - no amplification c -linear amplification e -exponential amplification and completely remove the abundant messages, there will still be a significant waste in sequencing the subtracted libraries as the common messages will already have been identified. D IFFERENTIAL D ISPLAY Differential display technology works by the amplification of the 3¢ terminal portions of mRNAs and the visualization of those fragments on a DNA sequencing gel (Liang and Pardee, 1992). The anchored oligo-dT primers define the 3¢ end of the RNA, and then a limited number of short arbitrary primers are used to amplify most of the mRNA in a cell (Figure 2.6). The separation of the amplified fragments by denaturing polyacrylamide gel electrophoresis allows direct side-by-side comparison of most of 3 6 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 RNA sample 1 RNA sample 2 Single strand cDNA template First strand cDNA synthesis Amplification using pairwise combinations of short primers 3 cycles of low stringency PCR 25 cycles of high stringency PCR Label included for vizualization of PCR products Electrophorese on acrylamide gel Expose gel to x-ray film Primer pairs RNA sample 1 2 1 2 1 2 1 2 1 2 1 2 a b c d e f FIGURE 2.6. Differential display. Each sample is treated in the same way. The first- strand cDNA is synthesized. This is then amplified with a series of short primers for 3 cycles at low stringency to account for the length of the primers. After the first 3 cycles the amplification is continued for a further 25 cycles at high stringency. In the amplification, label is included so that the products can be visualized. The products are then separated on a polyacrylamide gel, and the bands are compared. Differential bands can be excised and characterized further. the mRNAs. The polymorphic bands representing the differentially expressed sequences can be excised from the gel, reamplified, cloned, and sequenced. DNA M ICROARRAYS The combination of sequence information and automation has facilitated the change from looking at one gene at a time to being able to profile expression and changes of expression for many genes. DNA microarrays, which are one of the basic formats for looking at these changes, are orderly arrangements of DNA samples fabricated by high-speed robotics on glass, nylon, or other substrates. The initial experiments used cDNA immobilized on the surface with robot spotting, although a current alternative to the use of PCR prod- ucts is oligonucleotides either synthesized on the chip or synthesized off the chip and anchored later. These arrays can be used to detect polymorphisms and mutations, as well as to help map genomic libraries and characterize gene expression. The schema for a microarray experiment is shown in Figure 6.2. The steps involved are: 1. Development of the microaray. As mentioned above, this can be an array based on either fragments amplified through PCR or small oligonucleotides that are attached to the substrate. In either case, the sequences to be placed on the microarray must be selected. The PCR products, for example, can be amplified from cDNAs that comprise a unigene set developed from EST collections (see Chapter 4 and Figure 4.1) or from genomic DNA by using primers based on gene predic- tions from a genomic sequence. The oligonucleotides can be designed from the same information source. 2. The arrays are then printed by using the information. When the PCR products or the oligonucleotides are first generated and then attached to a substrate, this results in a more flexible platform because the array designed can be changed relatively easily and less expensively than when the oligonucleotides are actually synthesized on the substrate. 3. The probes are labeled and hybridized to the microarrays. The design of the experiment and the number of replicates are important para- meters for any differences observed to be statistically validated. 4. The hybridization data are analyzed with a series of software pro- grams, and any patterns of coordinated changes in gene expression are detected. 5. The form in which the data are reported is important for comparison between experiments. A series of standards for such reporting have C L O N I N G S Y S T E M S 3 7 been developed, and are continuing to be developed, and are known as the minimal information about a microarray experiment (MIAME; Brazma et al., 2001). Download 1.13 Mb. Do'stlaringiz bilan baham: |
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