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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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Agarose gel electrophoresis images from a) Algal ITS1 (∼100–300 bp), b) Algal ITS2 (∼300 bp) and c) Archaeal 16s (∼200 bp).Sample order: Lane 1: DNA ladder, 2: Modern Saline Valley, 3: Saline Valley ∼36 ka, 4: Saline Valley ∼64 ka, 5: Saline Valley ∼150 ka, and 6: PCR –ve.
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pone-0020683-g004: Agarose gel electrophoresis images from a) Algal ITS1 (∼100–300 bp), b) Algal ITS2 (∼300 bp) and c) Archaeal 16s (∼200 bp).Sample order: Lane 1: DNA ladder, 2: Modern Saline Valley, 3: Saline Valley ∼36 ka, 4: Saline Valley ∼64 ka, 5: Saline Valley ∼150 ka, and 6: PCR –ve.

Mentions: Modern and ancient halite crystals (36 ka, 64 ka, 150 ka, Figure 1) from Saline Valley were surface sterilized using the acid bleach AcBl protocol and DNA was extracted using the desalting protocol. The DNA extracts were screened using PCR with primers specific to Algal ITS1, ITS2 [23] and Archaeal 16s rDNA [24], [25]. All four samples showed positive amplification with expected fragment sizes for the primers used (Figure 4).


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)

Agarose gel electrophoresis images from a) Algal ITS1 (∼100–300 bp), b) Algal ITS2 (∼300 bp) and c) Archaeal 16s (∼200 bp).Sample order: Lane 1: DNA ladder, 2: Modern Saline Valley, 3: Saline Valley ∼36 ka, 4: Saline Valley ∼64 ka, 5: Saline Valley ∼150 ka, and 6: PCR –ve.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3111412&req=5

pone-0020683-g004: Agarose gel electrophoresis images from a) Algal ITS1 (∼100–300 bp), b) Algal ITS2 (∼300 bp) and c) Archaeal 16s (∼200 bp).Sample order: Lane 1: DNA ladder, 2: Modern Saline Valley, 3: Saline Valley ∼36 ka, 4: Saline Valley ∼64 ka, 5: Saline Valley ∼150 ka, and 6: PCR –ve.
Mentions: Modern and ancient halite crystals (36 ka, 64 ka, 150 ka, Figure 1) from Saline Valley were surface sterilized using the acid bleach AcBl protocol and DNA was extracted using the desalting protocol. The DNA extracts were screened using PCR with primers specific to Algal ITS1, ITS2 [23] and Archaeal 16s rDNA [24], [25]. All four samples showed positive amplification with expected fragment sizes for the primers used (Figure 4).

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