Nucleic Acid Extractions from Synthetic Mars Analog Soils

Complex soil matrices like those containing iron (III) oxides, for instance, have strong inhibitory effects towards nucleic acid extraction due to problems of competitive binding and the formation of destructive hydroxyl radicals. These soil-DNA interactions are especially problematic in low-biomass terrestrial environments and pose similar challenges for the extraction of nucleic acids from martian soils for in situ life detection via sequencing. 

We have developed methods of mitigating soil-DNA interactions which are capable of reliably extracting DNA from soils containing down to 10⁶ cells per gram of soil*. In addition, we have determined isolated DNA is of sufficient purity and does not contain carryover that may inhibit PCR and nanopore sequencing protocols.

*Our extractions from Mars analog soils (as described in Schuerger et al. 2012) are primarily conducted using Claremont BioSolution's PureLyse® Bacterial gDNA extraction kits and 50 mg of sample.

Mojarro et al. (2019) Astrobiology, PDF

Mojarro et al. (2017) Astrobiology, PDF

Mojarro et al. (2017) LPS XLVIII, PDF, ePoster

Mojarro et al. (2016). LPS XXXXVII, PDF, ePoster

Mojarro et al. (2017) Astrobiology, 2017

Mojarro et al. (2017) Astrobiology, 2017

Protocol

Note: This protocol is intended for low-biomass and recalcitrant in situ soil extractions, not for laboratory high-yield results.

Sample Prep (optional: for high-salt samples)

  1. Suspend your 50 mg sample in 800 µL of 8x CBIO binding buffer.

  2. Desalt your sample in a 100 kDA Amicon Ultra 0.5 column.

    • multiple washes may be required based on your salt concentration or waste color.

  3. Recover your sample in 400 µL of 8x CBIO binding buffer.

  4. Add ~4-6 µg of random hexamer primers (or any similar competitive binder/blocking agent) and mix your sample.

    • you may have to empirically determine what amount works best for your sample.

  5. Add 400 µL of water for a final 4x CBIO binding buffer solution.

  6. Skip to step 4 below.

Extraction

  1. Suspend your 50 mg sample in 400 µl of 8x CBIO binding buffer.

  2. Add ~4-6 µg of random hexamer primers (or any similar competitive binder/blocking agent) and mix your sample.

    • You may have to empirically determine what amount works best for your sample.

  3. Add 400 µL of water for a 4x CBIO binding buffer solution.

  4. Lyse for 2 minutes using your PureLyse® device @ 6 V (4x AAA batteries).

  5. Wash for 1 minute @ 1.5 V (3x dummy batteries + 1x AAA battery) by drawing 2.5 mL of 1x CBIO binding buffer into your PureLyse® device.

  6. Elute your DNA for 1 minute @ 1.5 V (3x dummy batteries + 1x AAA battery) using 200 µL of the CBIO elution buffer pre-heated to 70º C.

  7. Your eluted DNA is now ready for full genome amplification, 16S amplification, or low-input sequencing.