# Presentation: 018444

## DNA Extraction in Atacama Soils

*Lauren Fletcher – **NASA Ames Research Center/Stanford University*
*Christopher McKay – **NASA Ames Research Center*

- Spaceward Bound: Atacama Season 1

## Uranium P-36Explosive Space Modulator

- Top Secret

***Goal:  Venus Viewing Improvements***
***Plan:  Development of an Earth Shattering Explosive Device***

- Allied Tactical Attack Command
- Armed Martian Association

## Looking For MartiansIn the Atacama

*Lauren Fletcher – **NASA Ames Research Center/Stanford University*
*Christopher McKay – **NASA Ames Research Center*

- Spaceward Bound: Atacama Season 1

## A Little Bit about Mission Design

*Two General Laws Govern Mission Design:*
*Objectives*
*Exploration*
*Political*
*Planetary Defense*
*Science*
*Limitations*
*Monetary*
*Political*
*Physics*
*Technology*
*Science*
*Assume our Objective is the discovery of Life on Mars and that principle limitations are Scientific & Technology  *

## Stuff We Know About Mars

*We Know:*
*Conditions on early Mars are theorized to have been similar to that of Earth at the time of the genesis of life on Earth.*
**LONG* Periods when there were *LARGE* bodies of free flowing water on the Mars surface.*
*Continue to be periods when the conditions are right for to produce water vapor if not liquid water.*
*Large deposits of Subsurface and Polar Ice.*
*Thin Atmosphere.*
*We Conclude:*
*Life was at least *possible* on Mars*
*It is *still* possible that there is Life on Mars*
*Therefore, We Assume:*
*Life Does exist on Mars....we just haven’t found the right place or have had the right tool to find it.*

## Same Song, Second Verse(The Origin of Life on Earth/Mars)

*Life is the Same*
*Interchange of Planetary Materials*
*Extra-solar system meteorites hitting both planets.*
*Separate Genesis*
*Life is Different*
*Conditions on *both* planets acceptable for the initiation of Life, but a completely different genesis*
*Leads to Further Refinement of our Objective:*
*Is there Life?*
*Does it look like us?*
*Did we come from the same source or a separate Genesis?*

## The Complexity of Life on Mars

*The Answer is.....Not Very!*
*Current Scientific data shows a very stark environment devoid of all (VISIBLE) life.*
*Provides a Key Scientific Limitation:*
*Life on Mars is likely to be mono-cellular or not very complex multi-cellular organisms.*

*Therefore, We Conclude:*
*Looking for Microbes is the best way to find our Martians.*

## Direct and Indirect Detection of Martians

*Indirect – Looking for the *Signs* of Biology:*
*Respiration*
*Detection of Organic Material*
*Direct – We see it, therefore it is:*
*Cultivation*
*Microscopy*
*Direct Visual*
*Florescent Stain*
*Sublimation of Nucleic Acids*
*Phospholipid Fatty Acid Analysis*
*DNA Detection and Analysis*
*Comparison of the Methods used for the Direct Detection of Martians in the Atacama (following)*

## Slide 9

| Method | Reported Results | What does it really detect? | How related to
# bact/gm? | Sensitivity | Forms of Error |
| --- | --- | --- | --- | --- | --- |
| Cultiva-
tion | 10^4 CFU/gm
10^6 – 10^7 @ S25 ̊ | CFU/gm | Direct measurement of cultivatable heliotrophic bacteria/gram | >10^4 CFU/gm | Only cultivatable bacteria |
| QPCR | 166 bact/gm
peaks at 10^4 | Copies/gm | Direct measurement of all amplified copies of this 16sDNA sequence.  Divide # copies detected by 5.5 copies/bact. in order to convert to # bacteria/gm. | >9 bact/gm | DNA Shearing
Not a pan-generic primer
Assumed #copies/bact
% efficiency in removing DNA from soil
Interference of humic acid in amplification |
| DAPI | 10^6 – 10^7 bact/gm | Total Biomass by staining DAPI DNA
Bacteria determined during examination through microscope
Stains viable & nonviable only4 | Stains DAPI chain of DNA1, and stains total biomass.  The microscope operator is responsible for differentiating between bacterial and non-bacterial points. | Not Known.  No reference to sensitivity in publications read. | % efficiency in removing microbes from soil4
Results decrease significantly after 1-2 weeks of storage time1
Operator error in determining what constitutes a countable bacteria4
Methods of extrapolation of total bacteria from counts4
Masking of bacteria by soil particles |
| Sublima-
tion | 5x10^6 bact/gm
1 sample only | Total Biomas, by #gms Adenine/gm of soil to include viable, non-viable and dead (intact and non-intact) cells | Can’t be directly related.  No way to isolate bacteria from eukaryotes, and viruses | 5x10^6 | #gms per species is variable
70% of result could be dead2
Interference of  mineral surfaces, salts and larger amounts of organic matter in sublimation3
%efficiency of sublimation of Adenine from natural soils unknown3
Over-estimation due to eukaryotes and viruses.
Non-biologically produced Adenine present in sample |
| PLFA | To be Determined from last set of samples. | Total Biomas by #pmoles PLFA/gm of soil subdivided by composition |  | ~10^6 cells
or 50pmole of PLFA required for analysis.   | # of pmoles of PLFA/bact is variable
% efficiency in removing PLFA from soil-bound microbes. |

## Slide 10

| Method | Reported Results | What does it really detect? | How related to
# bact/gm? | Sensitivity | Forms of Error |
| --- | --- | --- | --- | --- | --- |
| Cultiva-
tion | 10^4 CFU/gm
10^6 – 10^7 @ S25 ̊ | CFU/gm | Direct measurement of cultivatable heliotrophic bacteria/gram | >10^4 CFU/gm | Only cultivatable bacteria |
| QPCR | 166 bact/gm
peaks at 10^4 | Copies/gm | Direct measurement of all amplified copies of this 16sDNA sequence.  Divide # copies detected by 5.5 copies/bact. in order to convert to # bacteria/gm. | >9 bact/gm | DNA Shearing
Not a pan-generic primer
Assumed #copies/bact
% efficiency in removing DNA from soil
Interference of humic acid in amplification |
| DAPI | 10^6 – 10^7 bact/gm | Total Biomass by staining DAPI DNA
Bacteria determined during examination through microscope
Stains viable & nonviable only4 | Stains DAPI chain of DNA1, and stains total biomass.  The microscope operator is responsible for differentiating between bacterial and non-bacterial points. | Not Known.  No reference to sensitivity in publications read. | % efficiency in removing microbes from soil4
Results decrease significantly after 1-2 weeks of storage time1
Operator error in determining what constitutes a countable bacteria4
Methods of extrapolation of total bacteria from counts4
Masking of bacteria by soil particles |
| Sublima-
tion | 5x10^6 bact/gm
1 sample only | Total Biomas, by #gms Adenine/gm of soil to include viable, non-viable and dead (intact and non-intact) cells | Can’t be directly related.  No way to isolate bacteria from eukaryotes, and viruses | 5x10^6 | #gms per species is variable
70% of result could be dead2
Interference of  mineral surfaces, salts and larger amounts of organic matter in sublimation3
%efficiency of sublimation of Adenine from natural soils unknown3
Over-estimation due to eukaryotes and viruses.
Non-biologically produced Adenine present in sample |
| PLFA | To be Determined from last set of samples. | Total Biomas by #pmoles PLFA/gm |  | ~10^6 cells
or 50pmole of PLFA required for analysis.   | # of pmoles of PLFA/bact is variable
% efficiency in removing PLFA from soil-bound microbes. |

## Slide 11

| Method | Reported Results | What does it really detect? | How related to
# bact/gm? | Sensitivity | Forms of Error |
| --- | --- | --- | --- | --- | --- |
| Cultiva-
tion | 10^4 CFU/gm
10^6 – 10^7 @ S25 ̊ | CFU/gm | Direct measurement of cultivatable heliotrophic bacteria/gram | >10^4 CFU/gm | Only cultivatable bacteria |
| QPCR | 166 bact/gm
peaks at 10^4 | Copies/gm | Direct measurement of all amplified copies of this 16sDNA sequence.  Divide # copies detected by 5.5 copies/bact. in order to convert to # bacteria/gm. | >9 bact/gm | DNA Shearing
Not a pan-generic primer
Assumed #copies/bact
% efficiency in removing DNA from soil
Interference of humic acid in amplification |
| DAPI | 10^6 – 10^7 bact/gm | Total Biomass by staining DAPI DNA
Bacteria determined during examination through microscope
Stains viable & nonviable only4 | Stains DAPI chain of DNA1, and stains total biomass.  The microscope operator is responsible for differentiating between bacterial and non-bacterial points. | Not Known.  No reference to sensitivity in publications read. | % efficiency in removing microbes from soil4
Results decrease significantly after 1-2 weeks of storage time1
Operator error in determining what constitutes a countable bacteria4
Methods of extrapolation of total bacteria from counts4
Masking of bacteria by soil particles |
| Sublima-
tion | 5x10^6 bact/gm
1 sample only | Total Biomas, by #gms Adenine/gm of soil to include viable, non-viable and dead (intact and non-intact) cells | Can’t be directly related.  No way to isolate bacteria from eukaryotes, and viruses | 5x10^6 | #gms per species is variable
70% of result could be dead2
Interference of  mineral surfaces, salts and larger amounts of organic matter in sublimation3
%efficiency of sublimation of Adenine from natural soils unknown3
Over-estimation due to eukaryotes and viruses.
Non-biologically produced Adenine present in sample |
| PLFA | To be Determined from last set of samples. | Total Biomas by #pmoles PLFA/gm of soil subdivided by composition |  | ~10^6 cells
or 50pmole of PLFA required for analysis.   | # of pmoles of PLFA/bact is variable
% efficiency in removing PLFA from soil-bound microbes. |

## Slide 12

| Method | Reported Results | What does it really detect? | How related to
# bact/gm? | Sensitivity | Forms of Error |
| --- | --- | --- | --- | --- | --- |
| Cultiva-
tion | 10^4 CFU/gm
10^6 – 10^7 @ S25 ̊ | CFU/gm | Direct measurement of cultivatable heliotrophic bacteria/gram | >10^4 CFU/gm | Only cultivatable bacteria |
| QPCR | 166 bact/gm
peaks at 10^4 | Copies/gm | Direct measurement of all amplified copies of this 16sDNA sequence.  Divide # copies detected by 5.5 copies/bact. in order to convert to # bacteria/gm. | >9 bact/gm | DNA Shearing
Not a pan-generic primer
Assumed #copies/bact
% efficiency in removing DNA from soil
Interference of humic acid in amplification |
| DAPI | 10^6 – 10^7 bact/gm | Total Biomass by staining DAPI DNA
Bacteria determined during examination through microscope
Stains viable & nonviable only4 | Stains DAPI chain of DNA1, and stains total biomass.  The microscope operator is responsible for differentiating between bacterial and non-bacterial points. | Not Known.  No reference to sensitivity in publications read. | % efficiency in removing microbes from soil4
Results decrease significantly after 1-2 weeks of storage time1
Operator error in determining what constitutes a countable bacteria4
Methods of extrapolation of total bacteria from counts4
Masking of bacteria by soil particles |
| Sublima-
tion | 5x10^6 bact/gm
1 sample only | Total Biomas, by #gms Adenine/gm of soil to include viable, non-viable and dead (intact and non-intact) cells | Can’t be directly related.  No way to isolate bacteria from eukaryotes, and viruses | 5x10^6 | #gms per species is variable
70% of result could be dead2
Interference of  mineral surfaces, salts and larger amounts of organic matter in sublimation3
%efficiency of sublimation of Adenine from natural soils unknown3
Over-estimation due to eukaryotes and viruses.
Non-biologically produced Adenine present in sample |
| PLFA | To be Determined from last set of samples. | Total Biomas by #pmoles PLFA/gm of soil subdivided by composition |  | ~10^6 cells
or 50pmole of PLFA required for analysis.   | # of pmoles of PLFA/bact is variable
% efficiency in removing PLFA from soil-bound microbes. |

## Slide 13

| Method | Reported Results | What does it really detect? | How related to
# bact/gm? | Sensitivity | Forms of Error |
| --- | --- | --- | --- | --- | --- |
| Cultiva-
tion | 10^4 CFU/gm
10^6 – 10^7 @ S25 ̊ | CFU/gm | Direct measurement of cultivatable heliotrophic bacteria/gram | >10^4 CFU/gm | Only cultivatable bacteria
(1-10% of Resident Population) |
| QPCR | 166 bact/gm
peaks at 10^4 | Copies/gm | Direct measurement of all amplified copies of this 16sDNA sequence.  Divide # copies detected by 5.5 copies/bact. in order to convert to # bacteria/gm. | >9 bact/gm | DNA Shearing
Not a pan-generic primer
Assumed #copies/bact
% efficiency in removing DNA from soil
Interference of humic acid in amplification |
| DAPI | 10^6 – 10^7 bact/gm | Total Biomass by staining DAPI DNA
Bacteria determined during examination through microscope
Stains viable & nonviable only4 | Stains DAPI chain of DNA1, and stains total biomass.  The microscope operator is responsible for differentiating between bacterial and non-bacterial points. | Not Known.  No reference to sensitivity in publications read. | % efficiency in removing microbes from soil4
Results decrease significantly after 1-2 weeks of storage time1
Operator error in determining what constitutes a countable bacteria4
Methods of extrapolation of total bacteria from counts4
Masking of bacteria by soil particles |
| Sublima-
tion | 5x10^6 bact/gm
1 sample only | Total Biomas, by #gms Adenine/gm of soil to include viable, non-viable and dead (intact and non-intact) cells | Can’t be directly related.  No way to isolate bacteria from eukaryotes, and viruses | 5x10^6 | #gms per species is variable
70% of result could be dead2
Interference of  mineral surfaces, salts and larger amounts of organic matter in sublimation3
%efficiency of sublimation of Adenine from natural soils unknown3
Over-estimation due to eukaryotes and viruses.
Non-biologically produced Adenine present in sample |
| PLFA | To be Determined from last set of samples. | Total Biomas by #pmoles PLFA/gm of soil subdivided by composition |  | ~10^6 cells
or 50pmole of PLFA required for analysis.   | # of pmoles of PLFA/bact is variable
% efficiency in removing PLFA from soil-bound microbes. |

## Summary

*There are also Advantages to Each Method:*
*Sensitivity*
*Ease of Sample Preparation*
*Have to Balance Advantages and Limitations in the selection of a specific method in order to meet your Objectives.*

## Proof of Martians on the Mars Surface

*The two recent Mars rovers have provided the proof of Martians on the Mars Surface!!!! *

## Martian on Mars Surface Photos

***Martian on Mars Surface Photos***

## The Secret Plot!!!

***The Secret Plot!!!***

*The two designers of the Optical Sensors Secretly Etched Two Martians on the Silicon Chip*
*There are four Martians on the Mars surface.*

## A Burning Man Duck-Mobile!

***A Burning Man Duck-Mobile!***