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United States Department of Agriculture

Agricultural Research Service

Title: Genome filtering using methylation-sensitive restriction enzymes with six-base pair recognition sites

Author
item Fellers, John

Submitted to: The Plant Genome
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 17, 2008
Publication Date: October 1, 2008
Citation: Fellers, J.P. 2008. Genome filtering using methylation-sensitive restriction enzymes with six-base pair recognition sites. The Plant Genome. 1:2.

Interpretive Summary: Genome sequencing is a means of finding all of the genes in an organism. Rice and Arabidopsis are two plant examples that have had all of their DNA sequenced. Unfortunately, some species have huge amounts of DNA. Wheat has 16 billion bases of DNA while rice only has 400 million, making wheat sequencing very expensive. Most of the wheat DNA, approximately 90%, consists of repeated sequences that do not code for genes. Sequencing the complete genome will be expensive not only in cost, but most of the sequence will not be informative. This manuscript is about a new technique that removes most of the repetitive DNA and enriches for gene-containing sequence. The technique uses enzymes that cut DNA, but only regions that have not been modified with a methyl molecule. The data presented shows that the technique is very efficient in wheat. It reduced the amount of repetitive DNA sequenced to as low as 16.2%. Other techniques can only reduce the repetitive DNA to just 30%. The technique was also effective in both corn and tobacco, which also have large genomes. By reducing the level of repeat DNA, it will be more cost effective to find the gene regions when doing genome sequencing.

Technical Abstract: 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 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 EST alignments, while tobacco libraries made with the same enzymes had 51.7% and 65.3%, respectively. This technique can be used with other methyl sensitive restriction filtration techniques to fill in gaps for assembling the gene space.

Last Modified: 12/21/2014
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