Genome Filtering Using Methylation- Sensitive Restriction Enzymes with Six Base Pair Recognition Sites

摘要

The large fraction of repetitive DNA in many plant genomes has complicated all aspects of DNA sequencing and assembly, and thus techniques that enrich for genes and low-copy sequences have been employed to isolate gene space. Methyl-sensitive restriction enzymes, with six base pair recognition sites, were evaluated on genomic DNA of the bread wheat ‘Chinese Spring’ as a different approach to enrich for genes. SacI, SalI, PstI, and AatII were used to digest wheat genomic DNA and fragments ranging from 400 bp to 2.0 kb were cloned and unidirectionally sequenced. All four enzymes provided some level of enrichment for gene space; however, AatII and PstI reduced the number of clones with repeat elements to just 16.2 and 19.1%, respectively. AatII and PstI were also effective in enrichment in corn and tobacco. Corn libraries made with AatII and PstI had 58.7 and 71.2%, respectively, of the clones with significant expressed sequence tag (EST) alignments, while tobacco libraries made with the same enzymes had 51.7 and 65.3%, respectively. With the development of ultra-throughput sequencing technologies, this technique provides an opportunity to rapidly and efficiently obtain sequencing from gene-rich regions.