Selective microbial genomic DNA isolation using restriction endonucleases.
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 Gm6AT...
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2014
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oai:doaj.org-article:f4589789518941dbb5e98d04134b80f02021-11-25T05:58:03ZSelective microbial genomic DNA isolation using restriction endonucleases.1932-620310.1371/journal.pone.0109061https://doaj.org/article/f4589789518941dbb5e98d04134b80f02014-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0109061https://doaj.org/toc/1932-6203To 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.Helen E BarnesGuohong LiuChristopher Q WestonPaula KingLong K PhamShannon WaltzKimberly T HelzerLaura DayDan SpharRobert T YamamotoR Allyn ForsythPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 10, p e109061 (2014) |
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| topic |
Medicine R Science Q |
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Medicine R Science Q Helen E Barnes Guohong Liu Christopher Q Weston Paula King Long K Pham Shannon Waltz Kimberly T Helzer Laura Day Dan Sphar Robert T Yamamoto R Allyn Forsyth Selective microbial genomic DNA isolation using restriction endonucleases. |
| description |
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. |
| format |
article |
| author |
Helen E Barnes Guohong Liu Christopher Q Weston Paula King Long K Pham Shannon Waltz Kimberly T Helzer Laura Day Dan Sphar Robert T Yamamoto R Allyn Forsyth |
| author_facet |
Helen E Barnes Guohong Liu Christopher Q Weston Paula King Long K Pham Shannon Waltz Kimberly T Helzer Laura Day Dan Sphar Robert T Yamamoto R Allyn Forsyth |
| author_sort |
Helen E Barnes |
| title |
Selective microbial genomic DNA isolation using restriction endonucleases. |
| title_short |
Selective microbial genomic DNA isolation using restriction endonucleases. |
| title_full |
Selective microbial genomic DNA isolation using restriction endonucleases. |
| title_fullStr |
Selective microbial genomic DNA isolation using restriction endonucleases. |
| title_full_unstemmed |
Selective microbial genomic DNA isolation using restriction endonucleases. |
| title_sort |
selective microbial genomic dna isolation using restriction endonucleases. |
| publisher |
Public Library of Science (PLoS) |
| publishDate |
2014 |
| url |
https://doaj.org/article/f4589789518941dbb5e98d04134b80f0 |
| work_keys_str_mv |
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| _version_ |
1718414378135453696 |