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Nucleic acid extraction yields with a standard and modified PureLyse® protocol for B. subtilis ATCC 6633 from water, 50 mg JSC Mars-1A, and 50 mg JSC 1A (n=3) . DNA Yield (%) is defined as DNA Out / DNA In *100.
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... subtilis spores processed with JSC 1A lowered DNA yields to 12.9% and JSC Mars-1A lowered yields to 3.23% (Figure 1). Decreased yields with JSC 1A may be associated with DNA adsorption to silicates within the regolith [22], considering that silicates are often utilized in DNA purification assays. ...
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... these additives are often inhibitory to downstream genomic analysis [19]. Instead we included random hexamer primers to JSC 1A, which increased DNA yields to 14.51% (Figure 1). These hexamers were removed from elutions with an extra cleaning step. ...
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... JSC Mars- 1A regolith, presumed DNA destruction by hydroxyl free-radicals was migitated by removing dissolved oxygen from the PureLyse® reagents through degas- sing in an anaerobic chamber. We observed a yield increase from 3.23% to 6.79% in JSC Mars-1A using this methodology (Figure 1). ...
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Citations
... Additional inhibition may emerge in the presence of silicates (Volossiouk et al., 1995;Melzak et al., 1996;Trevors, 1996), clay minerals (Greaves and Wilson, 1969), high concentrations of salt minerals, and acidic conditions (Henneberger et al., 2006). These soil-DNA interactions are especially problematic in low-biomass terrestrial environments (Barton et al., 2006;Azua-Bustos et al., 2012 and pose similar challenges for the extraction of nucleic acids from Mars analog soils (Navarro-Gonzalez et al., 2003;Mojarro et al., 2016). ...
We investigated the formation mechanisms of the nucleobases adenine and guanine and the nucleobase analogues hypoxanthine, xanthine, isoguanine, and 2,6-diaminopurine in a UV-irradiated mixed 10:1 H2O:NH3 ice seeded with precursor purine by using ab initio and density functional theory computations. Our quantum chemical investigations suggest that a multistep reaction mechanism involving purine cation, hydroxyl and amino radicals, together with water and ammonia, explains the experimentally obtained products in an independent study. The relative abundances of these products appear to largely follow from relative thermodynamic stabilities. The key role of the purine cation is likely to be the reason why purine is not functionalized in pure ammonia ice, where cations are promptly neutralized by free electrons from NH3 ionization. Amine group addition to purine is slightly favored over hydroxyl group attachment based on energetics, but hydroxyl is much more abundant due to higher abundance of H2O. The amino group is preferentially attached to the 6 position, giving 6-aminopurine, that is, adenine, while the hydroxyl group is preferentially attached to the 2 position, leading to 2-hydroxypurine. A second substitution by hydroxyl or amino group occurs at either the 6 or the 2 position depending on the first substitution. Given that H2O is far more abundant than NH3 in the experimentally studied ices (as well as based on interstellar abundances), xanthine and isoguanine are expected to be the most abundant bi-substituted photoproducts. Key Words: Astrophysical ice-Abiotic organic synthesis-Nucleic acids-Origin of life-RNA world. Astrobiology 17, xxx-xxx.
... Additional inhibition may emerge in the presence of silicates (Volossiouk et al., 1995;Melzak et al., 1996;Trevors, 1996), clay minerals (Greaves and Wilson, 1969), high concentrations of salt minerals, and acidic conditions (Henneberger et al., 2006). These soil-DNA interactions are especially problematic in low-biomass terrestrial environments (Barton et al., 2006;Azua-Bustos et al., 2012 and pose similar challenges for the extraction of nucleic acids from Mars analog soils (Navarro-Gonzalez et al., 2003;Mojarro et al., 2016). ...
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, xxx-xxx.
