WATER2g SOIL SAMPLEPREPREAGENT1SAMPLEPREPARATIONSONICATIONPREPREAGENT2FILTERANDPURIFYUSINGWASTEDNA-BINDING MATRIXMICROPROCESSORSAMPLESTORAGEBOARDMICROFLUIDICBUSHYDRAULICVALVESMULTIPLEPCRSTATIONSWITHHEATING/COOLINGMULTIPLEREAGENTANDFLUORESCENCESTORAGECHAMBERSOPTOELECTRONICSFORQUANTITATIVEPCRANDMELTCURVEANALYSISHYDRAULICLINEDEMULTIPLEXERMICROFLUIDICDEVICE
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CCD-based detection of SYBR GreenPDMSWellwithSYBRChipGreenlabeledDNAFluidicConnectionsExcitation FilterLEDThermal ControlMicro-channelplateDetection FilterCCDPeltierDevice
CCD-based detection of SYBR Green CCD Detection Filter Thermal Control Micro-channel plate LED Excitation Filter Fluidic Connections Well with SYBR Green labeled DNA PDMS Chip Peltier Device
Potential problemscommontoall lifedetectionexperiments1.LifeisextinctonMars.Some evidencethat PCR can detect extinct life.But Martian biota mayhave adapted to the gradual drying and cooling of Mars.PCR can detectspores which may survive for as long as 108 years.2.No lifeattheparticularlanding siteorintheparticular soil sampleanalysed.But PCR can detect one DNA molecule in a sample, i.e. a microbial cellthatblowsinfromanoasis3.Lifeexists much deeper in the soil, ie away fromthe UV and highoxidationpotentialonthesurface.PCRissensitiveenoughtodetectthedetritus(DNA)orsporesofsuch deep life.And we knowthat life thrives on redox gradients.4.NolifeonMars,neverwas
Potential problems common to all life detection experiments 1. Life is extinct on Mars. Some evidence that PCR can detect extinct life. But Martian biota may have adapted to the gradual drying and cooling of Mars. PCR can detect spores which may survive for as long as 108 years. 2. No life at the particular landing site or in the particular soil sample analysed. But PCR can detect one DNA molecule in a sample, i.e. a microbial cell that blows in from an oasis. 3. Life exists much deeper in the soil, ie away from the UV and high oxidation potential on the surface. PCR is sensitive enough to detect the detritus (DNA) or spores of such deep life. And we know that life thrives on redox gradients. 4. No life on Mars, never was
Complicationsto16SPCR1.ContaminationbyterrestrialmicrobesTreatreagentssothatcontaminationtakesmorethan4oPCRcyclestoappear.Window for detection of actual life on Mars will be PCR cycle 20to 40.Runtheinstrumentwithnosoil inputasa control.Hallmarkof insitulifewillbesignalstrengthscalingwithsoilinput.Expected to be more deeply branching in the tree of life, after 3.8 billion yearsofgenetic isolation.2.Martian16Sgenesarerelatedbuthavediverged fromtheEarthdefined universal motifsBroadenoursearch strategy to include16S primers that are a fewmutationsawayfromthecurrentlydefineduniversal.OruserandomprimerstoamplifyDNAthatisnot16Srelated---buthardtoclassifywhatcomesout.3.LifeonMarsisrelatedtolifeonEarthbuttransferredbeforetheevolutionof16SgeneComparisonofgenomesequencesmayallowthedetectionof moreancientuniversalgenes,andthedesignofuniversalprimers.4.LifeonMarsunrelatedtolifeonEarthWe are toast
Complications to 16S PCR 1. Contamination by terrestrial microbes Treat reagents so that contamination takes more than 40 PCR cycles to appear. Window for detection of actual life on Mars will be PCR cycle 20 to 40. Run the instrument with no soil input as a control. Hallmark of in situ life will be signal strength scaling with soil input. Expected to be more deeply branching in the tree of life, after 3.8 billion years of genetic isolation. 2. Martian 16S genes are related but have diverged from the Earth defined universal motifs Broaden our search strategy to include 16S primers that are a few mutations away from the currently defined universal. Or use random primers to amplify DNA that is not 16S related-but hard to classify what comes out. 3. Life on Mars is related to life on Earth but transferred before the evolution of 16S gene Comparison of genome sequences may allow the detection of more ancient universal genes, and the design of universal primers. 4. Life on Mars unrelated to life on Earth We are toast