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Ancient microbes from halite fluid inclusions: optimized surface sterilization and DNA extraction.

Sankaranarayanan K, Timofeeff MN, Spathis R, Lowenstein TK, Lum JK - PLoS ONE (2011)

Bottom Line: Fluid inclusions in evaporite minerals (halite, gypsum, etc.) potentially preserve genetic records of microbial diversity and changing environmental conditions of Earth's hydrosphere for nearly one billion years.Methods were verified on halite crystals of four different ages from Saline Valley, California (modern, 36 ka, 64 ka, and 150 ka), with retrieval of algal and archaeal DNA, and characterization of the algal community using ITS1 sequences.The protocol we developed opens up new avenues for study of ancient microbial ecosystems in fluid inclusions, understanding microbial evolution across geological time, and investigating the antiquity of life on earth and other parts of the solar system.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, State University of New York at Binghamton, Binghamton, New York, United States of America. ksankar1@binghamton.edu

ABSTRACT
Fluid inclusions in evaporite minerals (halite, gypsum, etc.) potentially preserve genetic records of microbial diversity and changing environmental conditions of Earth's hydrosphere for nearly one billion years. Here we describe a robust protocol for surface sterilization and retrieval of DNA from fluid inclusions in halite that, unlike previously published methods, guarantees removal of potentially contaminating surface-bound DNA. The protocol involves microscopic visualization of cell structures, deliberate surface contamination followed by surface sterilization with acid and bleach washes, and DNA extraction using Amicon centrifugal filters. Methods were verified on halite crystals of four different ages from Saline Valley, California (modern, 36 ka, 64 ka, and 150 ka), with retrieval of algal and archaeal DNA, and characterization of the algal community using ITS1 sequences. The protocol we developed opens up new avenues for study of ancient microbial ecosystems in fluid inclusions, understanding microbial evolution across geological time, and investigating the antiquity of life on earth and other parts of the solar system.

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Map showing location of Saline Valley, California and stratigraphic column of core SV-4A, showing uranium series ages (black arrows), sediment types, paleoenvironments, and sample depths (red arrows).Modified from Howe (1998) and Lowenstein et al. (2011) [34]. Upper right shows fluid filled inclusions in halite crystals from Saline Valley with algal forms trapped inside (arrows). a) Two algal cells (probably Dunaliella) in fluid inclusion in modern halite collected in April 2004. The red color likely comes from carotenoid pigments found in the cells. b) Fluid inclusion in halite crystal from core SV-4A (21.3 m depth, ∼36 ka), with algal cells and clumps of smaller biomaterials, probably prokaryotes. c) Fluid inclusion in halite from core SV-4A (43.9 m depth, ∼64 ka). Arrow shows whole cell; above arrow, cells are in various stages of degradation including the glycocalyx (cell coat) of several ruptured algal cells. d) Fluid inclusion in halite from core SV-4A (91.4 m depth, ∼150 ka), showing many well-preserved algal cells. Scale bars are 10 microns in all images.
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pone-0020683-g001: Map showing location of Saline Valley, California and stratigraphic column of core SV-4A, showing uranium series ages (black arrows), sediment types, paleoenvironments, and sample depths (red arrows).Modified from Howe (1998) and Lowenstein et al. (2011) [34]. Upper right shows fluid filled inclusions in halite crystals from Saline Valley with algal forms trapped inside (arrows). a) Two algal cells (probably Dunaliella) in fluid inclusion in modern halite collected in April 2004. The red color likely comes from carotenoid pigments found in the cells. b) Fluid inclusion in halite crystal from core SV-4A (21.3 m depth, ∼36 ka), with algal cells and clumps of smaller biomaterials, probably prokaryotes. c) Fluid inclusion in halite from core SV-4A (43.9 m depth, ∼64 ka). Arrow shows whole cell; above arrow, cells are in various stages of degradation including the glycocalyx (cell coat) of several ruptured algal cells. d) Fluid inclusion in halite from core SV-4A (91.4 m depth, ∼150 ka), showing many well-preserved algal cells. Scale bars are 10 microns in all images.

Mentions: Our study of the DNA trapped in fluid inclusions in ancient halite involves a six-stage protocol. (1) First, crystals with microscopically identified cells and cell structures are selected (Figure 1) to increase the likelihood of DNA retrieval. (2) Next, human DNA is spiked onto the crystal surface. (3) Spiked samples are then surface sterilized with the acid-bleach ‘AcBl’ protocol and (4) DNA is extracted using the desalting protocol. Surface sterilization is verified by the lack of amplification using human mitochondrial HV1 primers. (5) PCR amplification is used for targeted retrieval of DNA from different microbial kingdoms. (6) PCR products are cloned and sequenced to characterize microbial diversity to the genus level.


Ancient microbes from halite fluid inclusions: optimized surface sterilization and DNA extraction.

Sankaranarayanan K, Timofeeff MN, Spathis R, Lowenstein TK, Lum JK - PLoS ONE (2011)

Map showing location of Saline Valley, California and stratigraphic column of core SV-4A, showing uranium series ages (black arrows), sediment types, paleoenvironments, and sample depths (red arrows).Modified from Howe (1998) and Lowenstein et al. (2011) [34]. Upper right shows fluid filled inclusions in halite crystals from Saline Valley with algal forms trapped inside (arrows). a) Two algal cells (probably Dunaliella) in fluid inclusion in modern halite collected in April 2004. The red color likely comes from carotenoid pigments found in the cells. b) Fluid inclusion in halite crystal from core SV-4A (21.3 m depth, ∼36 ka), with algal cells and clumps of smaller biomaterials, probably prokaryotes. c) Fluid inclusion in halite from core SV-4A (43.9 m depth, ∼64 ka). Arrow shows whole cell; above arrow, cells are in various stages of degradation including the glycocalyx (cell coat) of several ruptured algal cells. d) Fluid inclusion in halite from core SV-4A (91.4 m depth, ∼150 ka), showing many well-preserved algal cells. Scale bars are 10 microns in all images.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3111412&req=5

pone-0020683-g001: Map showing location of Saline Valley, California and stratigraphic column of core SV-4A, showing uranium series ages (black arrows), sediment types, paleoenvironments, and sample depths (red arrows).Modified from Howe (1998) and Lowenstein et al. (2011) [34]. Upper right shows fluid filled inclusions in halite crystals from Saline Valley with algal forms trapped inside (arrows). a) Two algal cells (probably Dunaliella) in fluid inclusion in modern halite collected in April 2004. The red color likely comes from carotenoid pigments found in the cells. b) Fluid inclusion in halite crystal from core SV-4A (21.3 m depth, ∼36 ka), with algal cells and clumps of smaller biomaterials, probably prokaryotes. c) Fluid inclusion in halite from core SV-4A (43.9 m depth, ∼64 ka). Arrow shows whole cell; above arrow, cells are in various stages of degradation including the glycocalyx (cell coat) of several ruptured algal cells. d) Fluid inclusion in halite from core SV-4A (91.4 m depth, ∼150 ka), showing many well-preserved algal cells. Scale bars are 10 microns in all images.
Mentions: Our study of the DNA trapped in fluid inclusions in ancient halite involves a six-stage protocol. (1) First, crystals with microscopically identified cells and cell structures are selected (Figure 1) to increase the likelihood of DNA retrieval. (2) Next, human DNA is spiked onto the crystal surface. (3) Spiked samples are then surface sterilized with the acid-bleach ‘AcBl’ protocol and (4) DNA is extracted using the desalting protocol. Surface sterilization is verified by the lack of amplification using human mitochondrial HV1 primers. (5) PCR amplification is used for targeted retrieval of DNA from different microbial kingdoms. (6) PCR products are cloned and sequenced to characterize microbial diversity to the genus level.

Bottom Line: Fluid inclusions in evaporite minerals (halite, gypsum, etc.) potentially preserve genetic records of microbial diversity and changing environmental conditions of Earth's hydrosphere for nearly one billion years.Methods were verified on halite crystals of four different ages from Saline Valley, California (modern, 36 ka, 64 ka, and 150 ka), with retrieval of algal and archaeal DNA, and characterization of the algal community using ITS1 sequences.The protocol we developed opens up new avenues for study of ancient microbial ecosystems in fluid inclusions, understanding microbial evolution across geological time, and investigating the antiquity of life on earth and other parts of the solar system.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, State University of New York at Binghamton, Binghamton, New York, United States of America. ksankar1@binghamton.edu

ABSTRACT
Fluid inclusions in evaporite minerals (halite, gypsum, etc.) potentially preserve genetic records of microbial diversity and changing environmental conditions of Earth's hydrosphere for nearly one billion years. Here we describe a robust protocol for surface sterilization and retrieval of DNA from fluid inclusions in halite that, unlike previously published methods, guarantees removal of potentially contaminating surface-bound DNA. The protocol involves microscopic visualization of cell structures, deliberate surface contamination followed by surface sterilization with acid and bleach washes, and DNA extraction using Amicon centrifugal filters. Methods were verified on halite crystals of four different ages from Saline Valley, California (modern, 36 ka, 64 ka, and 150 ka), with retrieval of algal and archaeal DNA, and characterization of the algal community using ITS1 sequences. The protocol we developed opens up new avenues for study of ancient microbial ecosystems in fluid inclusions, understanding microbial evolution across geological time, and investigating the antiquity of life on earth and other parts of the solar system.

Show MeSH
Related in: MedlinePlus