RESUMO
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.
Assuntos
5-Metilcitosina/análise , Enzimas de Restrição do DNA , DNA Bacteriano/isolamento & purificação , DNA Fúngico/isolamento & purificação , DNA de Plantas/isolamento & purificação , DNA de Protozoário/isolamento & purificação , DNA Viral/isolamento & purificação , Desoxirribonuclease HpaII , Proteínas de Escherichia coli , Genética Microbiana/métodos , Genômica/métodos , Metagenoma , Ilhas de CpG/genética , Metilação de DNA , Enzimas de Restrição do DNA/isolamento & purificação , Enzimas de Restrição do DNA/metabolismo , DNA Bacteriano/genética , DNA Fúngico/genética , DNA de Plantas/genética , DNA de Protozoário/genética , DNA Viral/genética , Desoxirribonuclease HpaII/isolamento & purificação , Desoxirribonuclease HpaII/metabolismo , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/metabolismo , Biblioteca Gênica , Humanos , Microbiota/genética , Análise de Sequência de DNA , Escarro/microbiologia , Especificidade por SubstratoRESUMO
To improve the metagenomic analysis of complex microbiomes, we have repurposed restriction endonucleases as methyl specific DNA binding proteins. As an example, we use DpnI immobilized on magnetic beads. The ten minute extraction technique allows specific binding of genomes containing the DpnI Gm6ATC motif common in the genomic DNA of many bacteria including γ-proteobacteria. Using synthetic genome mixtures, we demonstrate 80% recovery of Escherichia coli genomic DNA even when only femtogram quantities are spiked into 10 µg of human DNA background. Binding is very specific with less than 0.5% of human DNA bound. Next Generation Sequencing of input and enriched synthetic mixtures results in over 100-fold enrichment of target genomes relative to human and plant DNA. We also show comparable enrichment when sequencing complex microbiomes such as those from creek water and human saliva. The technique can be broadened to other restriction enzymes allowing for the selective enrichment of trace and unculturable organisms from complex microbiomes and the stratification of organisms according to restriction enzyme enrichment.