*3.3. Detection of Nucleic Acids through Amplification Method*

Although a thermocycler instrument is not one of the pieces of equipment foreseen for the imminent exploration missions to Mars, it is a good candidate instrument to search for Earth-like life beyond Earth [40]. It is specific and sensitive, detecting even a single DNA molecule in a sample. The persistence of intact DNA has been tested on samples after a ground-based experiment simulating 16 months of exposure to space and Mars-like conditions outside the ISS. First, two different gene lengths of 939 bp and 330 bp, spanning the ribosomal LSU, have been targeted to be quantitatively amplified by qPCR based on the principle that damaged DNA is not amplifiable. Then, since the ribosomal genes occur in multiple copies in the genomes, we decided to amplify a region of 330 bp of the housekeeping gene β-actin, which is present in a single copy in the genome, to compare the level of detection based on the different gene length and number of copies in the genome.

In Figure 3, we reported the amplification of nucleic acids amplifying 939 bp and 330 bp of LSU gene and 330 bp of β-actin; the results showed a high amount of amplified DNA in all the experimental conditions tested and despite the gene type amplified: 13,170 DNA copies on average and never less than 102 copies were amplified. The test highlighted a common trend for all the samples, i.e., a lower amount of amplified DNA for samples exposed to UV radiation and vacuum (Top), and a higher copy number for samples exposed to vacuum but no radiation (Bottom) and in the control samples.

To conclude, all the gene amplifications worked out, and the number of amplified DNA copies was never under the amplification limit (one copy of a target sequence in genomic DNA), even when we decided to use the single copy gene β-actin.

**Figure 3.** (**A**) Quantitative PCR (qPCR) of a 939 bp target LSU (Large SubUnit) gene; (**B**) a 330 bp target gene (LSU) and (**C**) a 330 bp target gene (actin) of *C. antarcticus* DNA, after exposure to SVT treatments. On the axis of the ordinates the number of amplified copies on a logarithmic scale is shown; on the abscissa axis, the treatments are as follows: DNA from samples exposed to space simulated conditions (OS) and samples exposed to Mars-like conditions (P-MRS and S-MRS). OS = Original Substrate (sandstone); P-MRS = Phyllosilicatic Mars Regolith Stimulant; S-MRS = Sulfatic Mars Regolith Stimulant. Top (exposed to sun light with 0.1% Neutral Density filters), Bottom (dark control in space, not exposed to space radiation) and CTR (sample kept in the lab, in the dark at room temperature); POS CTR = DNA of *C. antarcticus* colony growth in physiological conditions (cultivated on MEA and incubated at 15 ◦C). The same letters above the bars indicate that the values are not statistically significant according to the *t* test (*p* ≤ 0.05). OS = Original Substrate, P-MRS = Phyllosilicatic Mars Regolith Simulant, S-MRS = Sulfatic Mars Regolith Simulant.
