stanford university dna extraction in atacama soils lauren fletcher – nasa ames research...
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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-36Explosive Space Modulator
Goal: Venus Viewing Improvements
Plan: Development of an Earth Shattering Explosive Device
Allied Tactical Attack CommandArmed Martian Association
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Looking For MartiansIn the Atacama
Lauren Fletcher – NASA Ames Research Center/Stanford University
Christopher McKay – NASA Ames Research Center
Spaceward Bound: Atacama Season 1
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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
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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.
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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?
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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.
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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)
Method Reported Results
What does it really detect?
How related to# bact/gm?
Sensitivity Forms of Error
Cultiva-tion
10^4 CFU/gm10^6 – 10^7 @ S25˚
CFU/gm Direct measurement of cultivatable heliotrophic bacteria/gram
>10^4 CFU/gm Only cultivatable bacteria
QPCR 166 bact/gmpeaks 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 ShearingNot a pan-generic primerAssumed #copies/bact% efficiency in removing DNA from soilInterference of humic acid in amplification
DAPI 10^6 – 10^7 bact/gm
Total Biomass by staining DAPI DNABacteria determined during examination through microscopeStains 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/gm1 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 variable70% 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 cellsor 50pmole of PLFA required for analysis.
# of pmoles of PLFA/bact is variable% efficiency in removing PLFA from soil-bound microbes.
Method Reported Results
What does it really detect?
How related to# bact/gm?
Sensitivity Forms of Error
Cultiva-tion
10^4 CFU/gm10^6 – 10^7 @ S25˚
CFU/gm Direct measurement of cultivatable heliotrophic bacteria/gram
>10^4 CFU/gm Only cultivatable bacteria
QPCR 166 bact/gmpeaks 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 ShearingNot a pan-generic primerAssumed #copies/bact% efficiency in removing DNA from soilInterference of humic acid in amplification
DAPI 10^6 – 10^7 bact/gm
Total Biomass by staining DAPI DNABacteria determined during examination through microscopeStains 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/gm1 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 variable70% 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 cellsor 50pmole of PLFA required for analysis.
# of pmoles of PLFA/bact is variable% efficiency in removing PLFA from soil-bound microbes.
Method Reported Results
What does it really detect?
How related to# bact/gm?
Sensitivity Forms of Error
Cultiva-tion
10^4 CFU/gm10^6 – 10^7 @ S25˚
CFU/gm Direct measurement of
cultivatable heliotrophic bacteria/gram
>10^4 CFU/gm Only cultivatable bacteria
QPCR 166 bact/gmpeaks 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 ShearingNot a pan-generic primerAssumed #copies/bact% efficiency in removing DNA from soilInterference of humic acid in amplification
DAPI 10^6 – 10^7 bact/gm
Total Biomass by staining DAPI DNABacteria determined during examination through microscopeStains 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/gm1 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 variable70% 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 cellsor 50pmole of PLFA required for analysis.
# of pmoles of PLFA/bact is variable% efficiency in removing PLFA from soil-bound microbes.
Method Reported Results
What does it really detect?
How related to# bact/gm?
Sensitivity Forms of Error
Cultiva-tion
10^4 CFU/gm10^6 – 10^7 @ S25˚
CFU/gm Direct measurement of cultivatable heliotrophic bacteria/gram
>10^4 CFU/gm
Only cultivatable bacteria
QPCR 166 bact/gmpeaks 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 ShearingNot a pan-generic primerAssumed #copies/bact% efficiency in removing DNA from soilInterference of humic acid in amplification
DAPI 10^6 – 10^7 bact/gm
Total Biomass by staining DAPI DNABacteria determined during examination through microscopeStains 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/gm1 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 variable70% 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 cellsor 50pmole of PLFA required for analysis.
# of pmoles of PLFA/bact is variable% efficiency in removing PLFA from soil-bound microbes.
Method Reported Results
What does it really detect?
How related to# bact/gm?
Sensitivity Forms of Error
Cultiva-tion
10^4 CFU/gm10^6 – 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/gmpeaks 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 ShearingNot a pan-generic primerAssumed #copies/bact% efficiency in removing DNA from soilInterference of humic acid in amplification
DAPI 10^6 – 10^7 bact/gm
Total Biomass by staining DAPI DNABacteria determined during examination through microscopeStains 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/gm1 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 variable70% 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 cellsor 50pmole of PLFA required for analysis.
# of pmoles of PLFA/bact is variable% efficiency in removing PLFA from soil-bound microbes.
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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.
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Proof of Martians on the Mars Surface The two recent Mars rovers have provided the
proof of Martians on the Mars Surface!!!!
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Martian on Mars Surface Photos
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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.
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A Burning Man Duck-Mobile!