"Frontmatter". In: Plant Genomics and Proteomics
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Christopher A. Cullis - Plant Genomics and Proteomics-J. Wiley & Sons (2004)
bidopsis can be accounted for by the relative amounts of methylated repeated
DNA. Thus the level of gene methylation may be about the same in the two species. However, using the methylation status will render some parts of the genome that are of interest inaccessible. Thus, for example, the methylation status of genes, or portions of genes, can differ markedly between tissues and/or developmental stages. Furthermore, the association between methy- lation and expression is certainly not absolute. Therefore, the choice of tissue from which the library is constructed is important (Yuan et al., 2002). The removal of methylated regions can be achieved at two different points in the cloning procedure. The first approach is with the use of a methylation-sensitive restriction enzyme, that is, one where the enzyme does not cleave the DNA if a cytosine in its recognition site is methylated. This approach has been used successfully to isolate low-copy-number sequences for molecular mapping probes, especially when using the enzyme PstI. The second method takes advantage of the E. coli pathway that digests incom- ing sequences if they are methylated. Mammalian and plant DNA can be cloned more efficiently in bacterial strains that are mutant at the mcrA and mcrBC loci because these strains do not eliminate recombinants that contain methylated cytosines. Most of the time the cloning strategy is to get a high representation of all the sequences present. By using E. coli that still have a functional mcr system, all those recombinants containing methylated DNA will be eliminated, leaving a library highly enriched in nonmethylated sequences. Therefore, with the known distribution of much of the methyla- tion in higher plants, the mcr system can be used to filter out methylated regions, resulting in an enrichment of genes at the expense of repeated sequences in such libraries. T RANSPOSON T AGS For some well-characterized genomes such as that of maize, certain families of transposable elements are known to have a preference to transpose into M E T H O D S O F F R A C T I O N AT I N G T H E G E N O M E 5 7 genes. Therefore, any method of isolating the regions adjacent to these ele- ments will also isolate sequences enriched in genes. However, it is unlikely that this could be extended to use as a general method for sequencing a gene set for most higher plants because the distribution and characterization of the transposon content has not been done. S ELECTING BAC C ONTIGS E NRICHED FOR E XPRESSED G ENES Rather than sequencing all the BACs that have been identified and placed on the physical map, those BAC contigs enriched for genes can be identified by using hybridization to overgo oligonucleotide probes (Han et al., 2000). Overgo probes are paired 24-mer oligonucleotides that contain an 8-bp complementary overlap. By annealing the oligonucleotides to each other and performing a [ 32 P]dCTP fill-in reaction, a labeled 40-mer is created that can be used for highly specific filter hybridization. This method has proven very efficient to construct sequence-ready BAC contigs for the human genome-sequencing project, where up to 40 distinct overgos have been used for simultaneous hybridization. The probes are designed from nonredundant cDNA or genomic sequences, but they still must be masked to eliminate repetitive elements. Therefore, some knowledge of the major families of repetitive elements for the species is necessary. The overgo probes are then designed from the masked sequence according to melting temper- ature, GC content, and potential for internal base pairing. The overgo probes are then hybridized to the BACs, and the labeled BACs are placed in the sequencing pipeline. Just sequencing the BACs containing expressed sequences will reduce the absolute amount of sequencing necessary. A pos- sible second benefit to this strategy would arise if there were gene-rich islands, because the BACs selected in this way would have a high density of genes on them. Download 1.13 Mb. Do'stlaringiz bilan baham: |
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