title: Selective microbial genomic DNA isolation using restriction endonucleases.
abstract: 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.
authors.affiliation: FLIR Systems, Inc., La Jolla, California, United States of America.
authors.forename: Helen E
authors.initials: HE
authors.lastname: Barnes
authors.affiliation: FLIR Systems, Inc., La Jolla, California, United States of America.
authors.forename: Guohong
authors.initials: G
authors.lastname: Liu
authors.affiliation: FLIR Systems, Inc., La Jolla, California, United States of America.
authors.forename: Christopher Q
authors.initials: CQ
authors.lastname: Weston
authors.affiliation: FLIR Systems, Inc., La Jolla, California, United States of America.
authors.forename: Paula
authors.initials: P
authors.lastname: King
authors.affiliation: FLIR Systems, Inc., La Jolla, California, United States of America.
authors.forename: Long K
authors.initials: LK
authors.lastname: Pham
authors.affiliation: FLIR Systems, Inc., La Jolla, California, United States of America.
authors.forename: Shannon
authors.initials: S
authors.lastname: Waltz
authors.affiliation: FLIR Systems, Inc., La Jolla, California, United States of America.
authors.forename: Kimberly T
authors.initials: KT
authors.lastname: Helzer
authors.affiliation: FLIR Systems, Inc., La Jolla, California, United States of America.
authors.forename: Laura
authors.initials: L
authors.lastname: Day
authors.affiliation: FLIR Systems, Inc., La Jolla, California, United States of America.
authors.forename: Dan
authors.initials: D
authors.lastname: Sphar
authors.affiliation: FLIR Systems, Inc., La Jolla, California, United States of America.
authors.forename: Robert T
authors.initials: RT
authors.lastname: Yamamoto
authors.affiliation: FLIR Systems, Inc., La Jolla, California, United States of America.
authors.forename: R Allyn
authors.initials: RA
authors.lastname: Forsyth
mesh_terms: D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics*; D016680:Genome, Bacterial; D023281:Genomics; D059014:High-Throughput Nucleotide Sequencing
chemical_list: D004269:DNA, Bacterial; D004262:DNA Restriction Enzymes