Selective microbial genomic DNA isolation using restriction endonucleases.

Autor: Barnes HE; FLIR Systems, Inc., La Jolla, California, United States of America., Liu G; FLIR Systems, Inc., La Jolla, California, United States of America., Weston CQ; FLIR Systems, Inc., La Jolla, California, United States of America., King P; FLIR Systems, Inc., La Jolla, California, United States of America., Pham LK; FLIR Systems, Inc., La Jolla, California, United States of America., Waltz S; FLIR Systems, Inc., La Jolla, California, United States of America., Helzer KT; FLIR Systems, Inc., La Jolla, California, United States of America., Day L; FLIR Systems, Inc., La Jolla, California, United States of America., Sphar D; FLIR Systems, Inc., La Jolla, California, United States of America., Yamamoto RT; FLIR Systems, Inc., La Jolla, California, United States of America., Forsyth RA; FLIR Systems, Inc., La Jolla, California, United States of America.
Jazyk: angličtina
Zdroj: PloS one [PLoS One] 2014 Oct 03; Vol. 9 (10), pp. e109061. Date of Electronic Publication: 2014 Oct 03 (Print Publication: 2014).
DOI: 10.1371/journal.pone.0109061
Abstrakt: 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.
Databáze: MEDLINE
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