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We describe continuing work to develop restriction endonucleases as tools to enrich targeted genomes of interest from diverse populations. Two approaches were developed in parallel to segregate genomic DNA based on cytosine methylation. First, the methyl-sensitive endonuclease HpaII was used to bind non-CG methylated DNA. Second, a truncated fragment of McrB was used to bind CpG methylated DNA. Enrichment levels of microbial genomes can exceed 100-fold with HpaII allowing improved genomic detection and coverage of otherwise trace microbial genomes from sputum. Additionally, we observe interesting enrichment results that correlate with the methylation states not only of bacteria, but of fungi, viruses, a protist and plants. The methods presented here offer promise for testing biological samples for pathogens and global analysis of population methylomes.

Epigenetic Segregation of Microbial Genomes from Complex Samples Using Restriction Endonucleases HpaII and McrB