Nucleic Acid Extraction from Synthetic Mars Analog Soils forin situLife Detection

Abstract

Biological informational polymers such as nucleic acids have the potential to provide unambiguous evidence of life beyond Earth. To this end, we are developing an automated in situ life-detection instrument that integrates nucleic acid extraction and nanopore sequencing: the Search for Extra-Terrestrial Genomes (SETG) instrument. Our goal is to isolate and determine the sequence of nucleic acids from extant or preserved life on Mars, if, for example, there is common ancestry to life on Mars and Earth. As is true of metagenomic analysis of terrestrial environmental samples, the SETG instrument must isolate nucleic acids from crude samples and then determine the DNA sequence of the unknown nucleic acids. Our initial DNA extraction experiments resulted in low to undetectable amounts of DNA due to soil chemistry–dependent soil-DNA interactions, namely adsorption to mineral surfaces, binding to divalent/trivalent cations, destruction by iron redox cycling, and acidic conditions. Subsequently, we developed soil-specific extraction protocols that increase DNA yields through a combination of desalting, utilization of competitive binders, and promotion of anaerobic conditions. Our results suggest that a combination of desalting and utilizing competitive binders may establish a “universal” nucleic acid extraction protocol suitable for analyzing samples from diverse soils on Mars. Key Words: Life-detection instruments—Nucleic acids—Mars—Panspermia. Astrobiology 17, 747–760.

Nucleic acid extraction and sequencing from low-biomass synthetic Mars analog soils for in situ life detection

“…The extractions from spore DNA in water and soils at 10 4 spores yielded similar results to our prior experiments at 10 8 spores used to develop the initial modified protocol (Mojarro et al, 2017b). We previously hypothesized that low DNA yields from B. subtilis was generally due to small acidsoluble proteins (SASPs) which bind to the DNA phosphate backbone and furnish protection from heat, salts, desiccation, and UV radiation in the spore state (Moeller et al, 2012(Moeller et al, , 2009).…”

“…In short, SASPs could inhibit interactions between the phosphate backbone and the Purelyse® oxide ceramic microbeads, which would ideally attract negatively charged polymers (i.e., DNA). In Mojarro et al, 2017b we seemingly validated this hypothesis by increasing spore DNA yields in water from 15% to 43% using a proof of concept protein separation binding buffer/phenol cocktail.…”

“…The lunar analog represents our unaltered soil control and is referred to as basalt. Details concerning the exact methods used to synthesize these soils can be found in Schuerger et al, 2012 andin Mojarro et al, 2017b.…”

“…Spore suspensions of Bacillus subtilis (ATCC 6633) similar to those used in clean-room sterilization effectiveness (Friedline et al, 2015) and bacteriostasis testing under Mars-like conditions (Kerney and Schuerger, 2011;Schuerger et al, 2017Schuerger et al, , 2003 were acquired from Crosstex (Part# SBS-08) to represent a worst case DNA extraction scenario of a tough-to-lyse organism. Spore suspension were DNAse-treated (New England Biolabs, M0303L) in order to remove any extracellular DNA and counted using a viable spore assay on lysogeny broth agar plates (Mojarro et al, 2017b). We assume a single genome copy per DNAse-treated spore of B. subtilis for calculating DNA yield (DNAout/DNAin).…”

“…Our goal for SETG is to be capable of analyzing a variety of environmental samples relevant to the search for life (related or otherwise to life on Earth) on Mars. However, complex soils, especially those containing iron oxides found on Mars (Bell et al, 2000), greatly inhibit nucleic acid extraction from cells due to competitive adsorption onto mineral surfaces (Hurt et al, 2014;Jiang et al, 2012) or destruction due to hydroxyl radicals (Gates, 2009;Imlay and Linn, 1988) during cell lysis (Mojarro et al, 2017b). Additional inhibition may occur in the presence of silicates (Melzak et al, 1996;Trevors, 1996), clays (Greaves and Wilson, 1969), and salts (Henneberger et al, 2006) due to adsorption and or DNA hydrolysis, denaturation and depurination at alkaline to acidic conditions (Gates, 2009).…”