Open AccessSelective Microbial Genomic DNA Isolation Using Restriction Endonucleases
Author(s) -
Helen Barnes,
Guohong Liu,
Christopher Q. Weston,
Paula King,
Long Pham,
Shan Waltz,
Kimberly T. Helzer,
Laura Day,
Dan Sphar,
R. T. Yamamoto,
R. Allyn Forsyth
Publication year - 2014
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0109061
Subject(s) - metagenomics , biology , dna , genomic dna , restriction enzyme , genome , microbiome , dna sequencing , computational biology , human genome , genetics , escherichia coli , dna extraction , genomic library , gene , polymerase chain reaction , base sequence
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 G m6 ATC 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.