title: Ancient microbes from halite fluid inclusions: optimized surface sterilization and DNA extraction.
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.
authors.affiliation: Department of Biological Sciences, State University of New York at Binghamton, Binghamton, New York, United States of America. ksankar1@binghamton.edu
authors.forename: Krithivasan
authors.initials: K
authors.lastname: Sankaranarayanan
authors.forename: Michael N
authors.initials: MN
authors.lastname: Timofeeff
authors.forename: Rita
authors.initials: R
authors.lastname: Spathis
authors.forename: Tim K
authors.initials: TK
authors.lastname: Lowenstein
authors.forename: J Koji
authors.initials: JK
authors.lastname: Lum
mesh_terms: D001105:Archaea / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D002140:California; D003460:Crystallization; D019641:DNA, Archaeal / Q000235:genetics / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D014644:Genetic Variation; D006801:Humans; D008903:Minerals / Q000737:chemistry*; D008969:Molecular Sequence Data; D010802:Phylogeny; D013242:Sterilization / Q000379:methods*; D013499:Surface Properties
chemical_list: D019641:DNA, Archaeal; D008903:Minerals