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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, Human HV1 PCR, Product size ∼440 bp.a) Evaluation of different surface sterilization protocols. Lane 1: DNA ladder, 2: Ac, 3:Al, 4:Bl, 5:AlBl, 6:AlAc, 7:AcBl, 8:AlAcBl, 9,12:Extract control, 10:Alcohol, 11:Spike, 13: PCR -ve. b) Effectiveness of surface sterilization. Halite crystals (modern Saline Valley) spiked with different amounts of human DNA. Lane 1: DNA ladder, Lanes 2,3,10,11: 1.5 ng spike, Lanes 4,5,12,13: 3.0 ng spike, Lanes 6,7,14,15: 4.5 ng spike, Lanes 8,9,16,17: no spike. Lanes 2–9: no surface sterilization. Lanes 10–17 surface sterilized.
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pone-0020683-g002: Agarose gel electrophoresis, Human HV1 PCR, Product size ∼440 bp.a) Evaluation of different surface sterilization protocols. Lane 1: DNA ladder, 2: Ac, 3:Al, 4:Bl, 5:AlBl, 6:AlAc, 7:AcBl, 8:AlAcBl, 9,12:Extract control, 10:Alcohol, 11:Spike, 13: PCR -ve. b) Effectiveness of surface sterilization. Halite crystals (modern Saline Valley) spiked with different amounts of human DNA. Lane 1: DNA ladder, Lanes 2,3,10,11: 1.5 ng spike, Lanes 4,5,12,13: 3.0 ng spike, Lanes 6,7,14,15: 4.5 ng spike, Lanes 8,9,16,17: no spike. Lanes 2–9: no surface sterilization. Lanes 10–17 surface sterilized.

Mentions: The effectiveness of 11 surface sterilization protocols (listed in Table 1) on modern halite crystals (Saline Valley 2004) was evaluated by testing for residual spiked human DNA following surface sterilization, using PCR primers targeting the HV1 region [21] of the human mitochondrion. The results (Figure 2a) indicate that several existing protocols including Vreeland et al. 2000 [16] (Figure 2a, Lane 6), and Fish et al. 2002 [12] (Figure 2a, Lane 10) fail to completely remove spiked human DNA (presence of HV1 PCR products). Among the other protocols tested, the addition of a bleach wash (6% sodium hypochlorite) to the Vreeland et al. 2000 [16] protocol (alkali-acid-bleach, “AlAcBl” on Table 1), or the use of an acid and bleach wash (“AcBl” on Table 1) proved sufficient in removing contaminating DNA (lack of HV1 PCR products, Figure 2a, Lanes 7, 8). A bleach wash was required to completely destroy surface DNA.


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, Human HV1 PCR, Product size ∼440 bp.a) Evaluation of different surface sterilization protocols. Lane 1: DNA ladder, 2: Ac, 3:Al, 4:Bl, 5:AlBl, 6:AlAc, 7:AcBl, 8:AlAcBl, 9,12:Extract control, 10:Alcohol, 11:Spike, 13: PCR -ve. b) Effectiveness of surface sterilization. Halite crystals (modern Saline Valley) spiked with different amounts of human DNA. Lane 1: DNA ladder, Lanes 2,3,10,11: 1.5 ng spike, Lanes 4,5,12,13: 3.0 ng spike, Lanes 6,7,14,15: 4.5 ng spike, Lanes 8,9,16,17: no spike. Lanes 2–9: no surface sterilization. Lanes 10–17 surface sterilized.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3111412&req=5

pone-0020683-g002: Agarose gel electrophoresis, Human HV1 PCR, Product size ∼440 bp.a) Evaluation of different surface sterilization protocols. Lane 1: DNA ladder, 2: Ac, 3:Al, 4:Bl, 5:AlBl, 6:AlAc, 7:AcBl, 8:AlAcBl, 9,12:Extract control, 10:Alcohol, 11:Spike, 13: PCR -ve. b) Effectiveness of surface sterilization. Halite crystals (modern Saline Valley) spiked with different amounts of human DNA. Lane 1: DNA ladder, Lanes 2,3,10,11: 1.5 ng spike, Lanes 4,5,12,13: 3.0 ng spike, Lanes 6,7,14,15: 4.5 ng spike, Lanes 8,9,16,17: no spike. Lanes 2–9: no surface sterilization. Lanes 10–17 surface sterilized.
Mentions: The effectiveness of 11 surface sterilization protocols (listed in Table 1) on modern halite crystals (Saline Valley 2004) was evaluated by testing for residual spiked human DNA following surface sterilization, using PCR primers targeting the HV1 region [21] of the human mitochondrion. The results (Figure 2a) indicate that several existing protocols including Vreeland et al. 2000 [16] (Figure 2a, Lane 6), and Fish et al. 2002 [12] (Figure 2a, Lane 10) fail to completely remove spiked human DNA (presence of HV1 PCR products). Among the other protocols tested, the addition of a bleach wash (6% sodium hypochlorite) to the Vreeland et al. 2000 [16] protocol (alkali-acid-bleach, “AlAcBl” on Table 1), or the use of an acid and bleach wash (“AcBl” on Table 1) proved sufficient in removing contaminating DNA (lack of HV1 PCR products, Figure 2a, Lanes 7, 8). A bleach wash was required to completely destroy surface DNA.

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