MolecularMicrobialEcologyManual

1.SimplifiedprotocolsforthepreparationofgenomicDNAfrombacterialcultures

2.ExtractionofribosomalRNAfrommicrobialcultures

3.ExtractionofmicrobialDNAfromaquaticsources:Marineenvironments

4.ExtractionofmicrobialDNAfromaquaticsources:Freshwater

5.MethodsforextractingDNAfrommicrobialmatsandcultivatedmicro-organisms:highmolecularweightDNAfromFrenchpresslysis

6.ExtractionofmicrobialDNAfromaquaticsediments

7.ExtractionofmicrobialRNAfromaquaticsources:Marineenvironments

8.ExtractionoftotalRNAandDNAfrombacterioplankton

9.MethodsforextractingRNAorribosomesfrommicrobialmatsandcultivatedmicroorganisms

10.Cellextractionmethod

11.DNAandRNAextractionfromsoil

12.RapidsimultaneousextractionofDNAandRNAfrombulkandrhizospheresoil

13.DirectExtractionofFungalDNAfromSoil

14.Purificationofmicrobialgenesfromsoilandrhizospherebymagneticcapturehybridizationandsubsequentamplificationoftargetgenes

byPCR

15.Directribosomeisolationfromsoil

16.DNAExtractionfromActinorhizalNodules

17.Quantificationofnucleicacids

18.Quantificationofnucleicacidsfromaquaticenvironmentsbyusinggreen-fluorescentdyesandmicrotiterplates

19.DegradationandturnoverofextracellularDNAinmarinesediments

20.IncorporationofthymidineintoDNAofsoilbacteria

21.Preparationofradioactiveprobes

22.DetectionofNucleicAcidsbyChemiluminescence

23.Parametersofnucleicacidhybridizationexperiments

24.DetectionandquantificationofmicrobialDNAsequencesinsoilbySouthern-anddot/slotblothybridization

25.DetectionofmicrobialDNAsequencesbycolonyhybridization

26.PolymerasechainreactionanalysisofsoilmicrobialDNA

27.Detectionofmicrobialnucleicacidsbypolymerasechainreactioninaquaticsamples

28.IsolationanddetectionofbacterialDNAsequencesindairyproducts

29.QuantitativePCRofenvironmentalsamples

30.Molecularbeaconsforhomogeneousreal-timemonitoringofamplificationproducts

31.DetectionandenumerationofsoilbacteriausingtheMPN-PCRtechnique

32.DetectionofmRNAandrRNAviareversetranscriptionandPCRinsoil

33.AmplificationofribosomalRNAsequences

34.Cloning16SrRNAgenesandutilizationtotypebacterialcommunities

35.SARST,SerialAnalysisofRibosomalSequenceTags

36.OligonucleotideFingerprintingofRibosomalRNAGenes(OFRG)

37.GenotypingofbacterialisolatesfromtheenvironmentusingLow-Molecular-WeightRNAfingerprints

38.Characterizationofthediversityofecologicallyimportantmicrobesbyrep-PCRgenomicfingerprinting

39.GenomicFingerprintingofMicro-organismsbyAFLP(TM)andERIC-anchorPCR

40.Theuseofpulsed-fieldgelelectrophoresistostudybacteriarecoveredfromtheenvironment

41.EasyindividualstrainandcommunitytypingbyrDNAITS1analysis

42.InsituPCRmethodologiesforvisualizationofmicroscalegeneticandtaxonomicdiversitiesofprokaryoticcommunities

43.Sensitivemulti-colorfluorescenceinsituhybridizationfortheidentificationofenvironmentalmicroorganisms

44.UseofClonedArtificialTargetsforFISH(catFISH)fortheoptimizationofoligonucleotideprobehybridizationconditionswith16SrRNA

clonesforinsituquantificationofuncultivatedprokaryoticcells

45.Denaturinggradientgelelectrophoresis(DGGE)inmicrobialecology

46.FungalCommunityAnalysisusingPCR-DenaturingGradientGelElectrophoresis(DGGE)

47.TheAnalysisofMicrobialCommunitieswithTerminalRestrictionFragmentLengthPolymorphism(T-RFLP)

48.MicrobialcommunityanalysisbyPCR-single-strandconformationpolymorphism(PCR-SSCP)

49.IsolationofhighmolecularweightgenomicDNAfromsoilbacteriaforgenomiclibraryconstruction

50.UseofBiolog(R)fortheCommunityLevelPhysiologicalProfiling(CLPP)ofenvironmentalsamples

51.Auorescentstainingofmicrobesfortotaldirectcounts

52.DetectionofmicrobesbyScanningConfocalLaserMicroscopy(SCLM)

53.Productionofanti-microbialantibodiesandtheirutilizationinstudiesofmicrobialautecologybyimmunofluorescencemicroscopyand

insituCMEIASimageanalysis

54.Theslideimmunoenzymaticassay(SIA):Asimpleandlowcostsystemsuitablefordetectingwater-bornemicrobeswithouttheneed

forsophisticatedtechnologicalinfrastructure

55.InsituhybridizationtodetectmicrobialmessengerRNAinplanttissues

56.Fattyacidanalysisintheidentification,taxonomyandecologyof(plantpathogenic)bacteria

57.Determinationofmicrobialcommunitystructureusingphospholipidfattyacidprofiles

58.Respiratorylipoquinonesasbiomarkers

59.EnvironmentalProteomics:MethodsandApplicationsforAquaticEcosystems

60.Naturaltransformationinaquaticenvironments

61.Naturaltransformationinsoil:microcosmstudies

62.PlasmidTransferinAquaticEnvironments

63.Conjugationintheepilithon

64.Detectionofbacterialconjugationinsoil

65.Transductionintheaquaticenvironment

66.Phageecologyandgeneticexchangeinsoil

67.Lacasamarkergenetotrackmicrobesintheenvironment

68.XylEasamarkergeneformicroorganisms

69.GUSasamarkertotrackmicrobes

70.ThecelBmarkergene

71.Visualisationofmicrobesandtheirinteractionsintherhizosphereusingautofluorescentproteinsasmarkers

72.Identificationofbacteriabytheirintrinsicsequences:Probedesignandtestingoftheirspecificity

73.SubtractionhybridizationfortheproductionofhighspecificityDNAprobes

74.Considerationsfortheuseoffunctionalmarkersandfieldreleaseofgeneticallyengineeredmicroorganismstosoilsandplants

75.Applicationofecologicaldiversitystatisticsinmicrobialecology

76.Samplingefficiencyandinterpretationofdiversityin16SrRNAgenelibraries

77.LIBSHUFFComparisonsof16SrRNAGeneCloneLibraries

78.Clusteranalysisandstatisticalcomparisonofmolecularcommunityprofiledata

79.Computer-assistedanalysisofmolecularfingerprintprofilesanddatabaseconstruction

80.Multivariatestatisticalmethodsandartificialneuralnetworksforanalysisofmicrobialcommunitymolecularfingerprints

81.Quantitativefluorescenceinsituhybridisation(FISH):statisticalmethodsforvalidcellcounting

82.Oligonucleotideprobedesignformixedmicrobialcommunitymicroarraysandotherapplicationsandimportantconsiderationsfordata

analysis

83.Designofmicroarraysforgenome-wideexpressionprofiling

84.Assessmentofthemembranepotential,intracellularpHandrespirationofbacteriaemployingfluorescencetechniques

85.Useofmicroelectrodestomeasureinsitumicrobialactivitiesinbiofilms,sediments,andmicrobialmats

86.Applicationofwhole-cellbiosensorsinsoil

87.Detectionofbacterialhomoserinelactonequorumsensingsignals

88.BrdUSubstrateUtilizationAssay

89.Stableisotopeprobingofnucleicacidstoidentifyactivemicrobialpopulations

90.Linkingmicrobialcommunitystructureandfunctioning:stableisotope(13C)labelingincombinationwithPLFAanalysis

91.Correlatingsingle-cellcountwithfunctioninmixednaturalmicrobialcommunitiesthroughSTARFISH

92.DifferentialdisplayofmRNA

93.Macro-arraysprotocolsforgeneexpressionstudiesinbacteria

94.Oligonucleotide-basedfunctionalgenearraysforanalysisofmicrobialcommunitiesintheenvironment

95.ProteomicAnalysisofBacterialSystems

MolecularMicrobialEcologyManual

KluwerAcademicPublishers2004

10.1007/l-4020-2177-l_l

Section1-IsolationofNucleicAcids

Simplifiedprotocolsforthepreparationof

genomicDNAfrombacterialcultures

EdwardMoore1,AngelikaArnscheidt1,AnnetteKrtJger1,

CarstenStrOmpl1andMargitMau1

(1)DivisionofMicrobiology,GBF-GermanNationalResearchCentreforBiotechnology,

MascheroderWeg1,D-38124,Braunschweig,Germany

Introduction

Thedevelopmentofmethodologiesfortheanalysisofmicroorganismsand

microbialecology,atthemolecularlevel(i.e.,nucleicacids,proteins,

lipids,andtheirgenes),hasprogressedphenomenallyinrecentyears.

Eachmethodologyhasspecificadvantagesanddisadvantages,or

complications.However,theadvancesinPCR,cloning,geneprobing,

sequencingandfingerprintinghaveenabledtechniquesexploitingnucleic

acidstobeutilisedextensivelyfortheanalysisofmicroorganisms.Often,

suchprotocolsrequire,firstly,thatthenucleicacidsareextractedin

aformwhichcanbeemployedfortheanalyses.Thismay,insomecases,

bemoredifficultthananticipatedinitially,sincemanybacteriaare

extremelyresistanttocelldisruption.Typically,theseare

Gram-positivebacteria(e.g.,Mycobacteriunispp.,Peptococcusspp.,

Rhodococcusspp.,etc.),aswellassomeArchae(e.g.,methanogens),with

thickcellwallsofpolysaccharideorpseudopeptidoglycan,andmany

speciesoffungiandalgae.

Generalconsiderations

Severalprotocolshavebeendevelopedanddescribedforthepreparation

ofgenomicDNAfrombacteria,beginningwiththeprototypalmethodof

Marrnur[16],whichinvolved:a)celldisruptionbyanenzyme-detergent

lysis;b)extractionswithorganicsolvents;andc)recoveryoftheDNA

byalcoholprecipitation.Subsequentprotocolshaveusuallyinvolvedsome

modificationofoneormoreofthesegeneralsteps.

Celldisruption

ThemostdifficultanduncertainstepinobtainingDNAfrombacterial

culturesisthatofdisruptingthecells.Thedifficultiesderive,inpart,

fromimposedlimitationsinthehandlingofthepreparations,whichare

necessaryforobtaininggenomicDNAofhighmolecularweight.Thus,in

general,themostdesirablemeansofdisruptingbacterialcellsfor

obtaininggenomicDNAisthroughenzymaticdigestionanddetergentlysis.

Suchastrategyisenhancedbypriortreatmentofcellswithametal

chelatingagent,suchasethylenediamine-tetraaceticacid(EDTA).Ifthe

cellwalloftheorganismissusceptibletosuchtreatments,relatively

highmo1ecu1ar-weightgenomicDNAcanbeobtainedwhichisapplicablefor

anumberofanalyticaltechniques.Further,thelysisshouldbecarried

outinabuffered(pH8-9)mediumcontainingEDTA.ThealkalinepHreduces

electrostaticinteractionsbetweenDNAandbasicproteins,assistsin

denaturingothercellularproteinsandinhibitsnucleaseactivities.EDTA

bindsdivalentcations,particularlyMg2andMn2',reducingthe

stabilitiesofthewallsandmembranesandalsoinhibitsnucleaseswhich

havearequirementformetalcations.

Celldisruptionbyenzymatictreatments

Lysozyme,isolatedcommerciallyfromchickeneggwhite,isamemberof

thebroadclassofmuramidaseswhichcatalysethehydrolysisofthe

P-1,4-glycosidiclinkagebetweentheN-acetylmuramic

acid-N-acetylglucosaminerepeatingunit,comprisingamajorpartofthe

peptidoglycanlayerofthecellwallsofmostbacteriaLysozymeis

especiallyeffectiveindisruptingbacterialcellswhenusedin

combinationwithEDTA[J5].Lysozymeandrelatedenyzmesareusefulfor

disruptingthecellsofabroadrangeofbacterialspecies,althoughmany

speciesarenotparticularlysusceptibletomuramidasetreatmentdue,

presumably,tolayersofproteinorcapsularslime,whichprotectthe

peptidoglycan.Additionally,astheircellwallsdonotcontain

peptidoglycan,alldescribedspeciesofArchaeareresistanttolysozyme

activity.

ProteinaseK,aserineproteaseproducedbythefungusTritirachiumalbum,

cleavesadjacenttothecarboxylgroupsofaliphaticandaromaticamino

acidsinvolvedinpeptidebonding\_4\,includingthosecomprisingthe

peptidecross-linkinginterbridgesofthepeptidoglycanlayersofthe

cellwallsofbacteria.TheapplicabilityofProteinaseKfordisrupting

bacterialcellwallsisenhancedbyitsinsensitivitytospecific

chelatingagents,allowingittobeutilisedincombinationwithEDTAand

lysozyme.However,thepeptideinterbridgesofthecellwallsofdifferent

species,formedbydifferentcombinationsofcomponentaminoacids,with

inherentlydifferentsusceptibilitiestocleavage,maybemoreorless

resistanttoProteinaseKlysis.

WhilelysozymeandproteinaseKare,probably,theenzymesmostcommonly

usedforthedisruptionofbacterialcells,additionalbacterial

cell-disruptingenzymesalsohavebeenreportedwithbroadornarrow

specificities.Othermuramidases,mutanolysinandlysostaphinreact,

analogoustolysozyme,atthepeptidelinkagesinthecellwalls,although

thespecieswhicharesusceptibletotheseenzymesdifferfromthosewhich

areaffectedbylysozyme[幺20,26].Subtilisinsareextracellular

proteases,producedbyBacillusspp.,exhibitingabroadspecificityin

hydrolysingmostpeptideandesterbonds\_24].Theyarenotinactivated

bychelatingagents,whichmakesthemapplicableincombinationwithEDTA.

Theapplicationofachromopeptidasehasbeenlimitedtothedisruption

ofGram-positivecells,principallystaphylococci[9],although

applicationswithotherbacteriahavebeenreported.

Celldisruptionbydetergenttreatments

Detergentsprovideeffective,yetrelativelygentle,meansfordisrupting

cells,bindingstronglytoproteinsandcausingirreversibledenaturation.

Further,conditionswhichcausedissociationofprotein(i.e.,highpH,

lowandhighionicstrength,etc.)tendtoenhance,aswell,the

solubilisationefficienciesofdetergents\_7].Detergentsare

particularlyeffectivefordisruptingbacteriawhentheircellwallshave

beendamaged(e.g.,throughtheactionsofmetalchelatingagents,

lysozymeandProteinaseK)priortotheiradditiontothecellsuspension.

Sodiumdodecylsulfate(SDS)isananionicdetergentwhichreacts,atlow

concentrations,atproteinhydrophobicsites,bindingcellularproteins

andlipoproteins,formingSDS-polypeptidemicellarcomplexes,and

effectivelydenaturingthemandpromotingthedissociationofnucleic

acids[77].Further,SDSinhibitsnucleasesanddoesnotinteractwith

thehydrophilicnucleicacids.SomeproteinsformSDScomplexesonlyafter

theyhavebeenheatedortreatedwithreagents(e.g.,mercaptoethanol)

tocleaveintraproteindisulfidebonds.

N-lauroylsarcosine(Sarcosyl),empirically,maybemoreeffectiveat

denaturingcellularpolysaccharidematerialandcanbeused,insteadof

SDS,forthedisruptionofbacterialcells(e.g.,Azotobacter,

Beijerinckia,Klebsiella,etc.)whichproducecopiousamountsofcapsule.

Cetyltrimethylammoniumbromide(CTAB),acationicdetergent,hasbeen

usedextensivelyinthepreparationofnucleicacidsfromfungiandplants,

whenlargeamountsofpolysaccharidematerialstendtointerferewiththe

extraction.However,CTABalsohasbeenprovenusefulforDNAextractions

frombacterialcellsbydenaturingandprecipitatingthecellwall

1ipopo1ysaccharidesandproteinsInthepresenceofmonovalent

cation(e.g.,Na")concentrationsabove0.5M,DNAwillremainsoluble.

Nonpolardetergents,includingtheTritonXseries,Tweenseries,Nonidet

P-40,etc.,aregenerally"milder”solubilisingagentsthanthepolar

detergentsandtheyseemtohaveamuchmorelimitedabilitytoinitiate

thedisruptionofbacterialcells.

Celldisruptionby“physical”methods

Bacteriawhosecellwallsarenotsusceptibletoenzymaticanddetergent

treatmentsmaybedisruptedusing“harsher”(i.e.,alsoontheDNA)

methodswhichmaybedescribed,arbitrarily,as"physical”or

“mechanical”[10,77,14,19\.SuchmethodsgenerateDNAwhichisoften

shearedandusuallynotoftherelativelyuniform,large,molecularweight

thatcanbeattainedusingenzymaticanddetergentdisruption.Thus,such

methodsmaynotbeappropriateforpreparingDNAforspecificanalytical

techniques.However,ininstanceswhereinithasnotbeencriticalthat

theDNAbeofuniformhighmolecularweight,methodsemployingaFrench

pressurecellorasonicatorhavebeenusedwithsuccess.Theuseofglass

particleswiththe(mini)-beadbeaterisparticularlyeffectivefor

disruptingmostbacteriaandisthemethodofchoiceforthepreparation

ofDNAfrombacterialcellsinproblematicmatrices(e.g.,soils)\_23\.

Additionally,amethodfortheproductionofhighmolecularweightDNA

fromGram-positiveandacid-fastbacteriausingamicrowaveovenhasbeen

described[7].However,theefficaciesofsuchmethods,allofwhich

requireadditional,specialised,equipment,havebeenlimited,inmost

cases,intherangeofbacteriaforwhichagivenmethodcanbeapplied.

Afurtherapplicationwhichhasbeenshowntobeeffective,particularly

incombinationwithothersteps,fordisruptingextremelyrecalcitrant

bacteriaisthefreeze(inliquidnitrogen)andfastthaw(at95-98°C)

technique.Thismethodisoftenusedinproceduresforextractingnucleic

acidsdirectlyfromenvironmentalsamples,suchassoilandsediment[22].

Suchatreatmentenhancesbacterialcelldisruption(e.g.,particularly

speciesproducingprotectivecapsularslimeandthoseinvolvedinthe

formationofbiofilms)byinducingphasechangesincellmembranesthrough

successive,rapid,extremesintemperaturewhichrendercellsmore

susceptibletoenzymaticanddetergentlysis.

Nucleicacidextractions

TheisolationofDNAfromcells(i.e.,selectivelyeliminatingother

cellularcomponentsexcepttheDNA)isthemoststraightforwardofthe

threegeneralsteps.Themethodsofchoiceforextractions,traditionally,

haveinvolvedtheapplicationoforganicsolvents(e.g.,phenoland

chloroform)[13],whichinteractwithhydrophobiccomponentsofprotein

andlipoprotein,causingdenaturation.Itisbelievedthatforces

maintainingthehydrophobicinteriorsofproteins,throughtheirnative

conformations,areovercomebyexposuretohydrophobicsolvents,

resultingintheunfoldingandprecipitationoftheprotein[6].The

precipitateofdenaturedcellularmaterialremainswithintheorganic

phase,whichisseparatedbycentrifugation.

Ingeneral,phenolisaneffectivedenaturingagentofprotein,while

chloroformwillbemoreeffectiveforpolysaccharidematerials.Thus,for

theextractionofDNAfrombacterialcells,mixturesofphenol/chloroform

aremoreeffectivethaneitheris,alone.Phenolofhighpurity(i.e.,

redistilled),saturatedandequilibratedwithbuffer(pH8)shouldbeused

fortheextractions.

RecoveryofDNA

ThestandardmethodforrecoveringDNAfromcelllysatesandsuspensions

isbytheuseofalcohol(i.e.,ethanolorisopropanol)reversible

denaturation(i.e.,thehelicalstructureisextensivelydestroyed)and

subsequentprecipitation[5],followedbycentrifugation.Itis

recognisedthatDNAprecipitatespoorlyinsalt-freesolutionsandthat

alcoholprecipitationsshouldbeperformedinthepresenceofamonovalent

cationwithaconcentrationof,atleast,0.1M.PrecipitationofDNAin

suspensionsisinitiatedbyadding0.1suspensionvolumeof3Msodium

acetate(pH5.2)and2.0-3.0suspensionvolumes(calculatedafterthe

additionofsalt)of100%ethanol(forDNAsuspensionsoflow

concentration,ahigherratioofethanoltosuspensionvolumewill

facilitateDNAprecipitation)\_2f\.Alternatively,0.5volumesof7.5M

ammoniumacetate(pH8)canbeusedinsteadofsodiumacetate[5].Inthis

case,smallnucleicacidfragments(approximately150nucleotidesand

smaller),willnotbeprecipitated,whichmaybeadvantageousinsome

cases.Isopropanol(0.5-1.0volumes)maybeused,ratherthanethanol,

particularlywhensmallvolumes(e.g.,lessthan1.0ml)areneeded.

AlthoughithasbecomeanacceptedpracticetocarryoutDNA

precipitationsatextremecoldtemperatures(e.g.,-70°C),datasuggest

thatprecipitationsatsuchtemperaturespresentnosignificantadvantage

overprecipitationscarriedoutinicewater(i.e.,approximately0°C)

and,infact,maybecounterproductive\_27\(Fig.1).Further,whilethe

majorityofDNAinconcentratedsuspensionsisrecoveredquickly(i.e.,

within5minutes)bycentrifugation(12,000-15,000Xg),therecovery

ofDNAfromdilutesuspensionsmayrequirecentrifugationsforaslong

as30minutes(Fig.2).

o

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6.

x

O8o

O

O

CM7o

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％(

)6o

5o

8

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-700

Temperature(℃)

Figure1TherecoveryofDNAasafunctionoftheprecipitationtemperature.Precipitationsof

varyingamounts(0.6ng-010pg)ofDNAatextremlylowtemperatures(i.e.,-70℃)areless

efficientthanat0℃.Theefficienciesofrecovery,bycentrifugation(12,000xg,6℃),were

alsoobservedtobedependentupontheamountsofDNAinsuspension.Thevaluesindicated

inthegraphrepresentthemeans,calculatedfromtheobservedrecoveriesfromsuspension,

ofvaryingamountsofDNA.Therangesofobservedrecoveriesareindicated,withthelowest

andhighestrecoveries,ateachtemperaturetested,andcorrespondtothelowestandhighest

concentrationsofDNA,respectively.ThegraphwaspreparedfromdatatakenfromZeugin

andHartley,1985\27\.

51015202530

Centrifugationtime(min)

Figure2TherecoveryofDNAasafunctionofthecentrifugationtime.Therecoveryofvarying

amounts(0.6ng-10pg)ofDNAisenhancedbyincreasedcentrifugationtimes.The

efficienciesofrecovery,bycentrifugation(12,000xg,6℃),werealsoobservedtobe

dependentupontheamountsofDNAinsuspension.Thevaluesindicatedinthegraph

representthemeans,calculatedfromtheobservedrecoveriesfromsuspension,ofvarying

amountsofDNA.Therangesofobservedrecoveriesareindicated,withthelowestandhighest

recoveries,ateachcentrifugationtimetested,andcorrespondto.thelowestandhighest

concentrationsofDNA,respectively.ThegraphwaspreparedfromdatatakenfromZeugm

andHartley,1985\27\.

Animportantconsiderationtokeepinmindthroughouttheextraction

processistherelationshipbetweentheamountofDNAinsuspensionand

theability,ultimately,torecoverit.

InstudiestodeterminetheoptimalconditionsfortherecoveryofDNA

fromsuspensionsbyprecipitationandcentrifugation[2刁,theamountof

DNArecoveredwasobservedtobeproportionaltotheconcentrationin

suspension(Fig.3).Thus,itisimportanttoconsiderthisrelationship

whendecidingupontheextractionprotocoltouseandsubsequenthandling

oftheDNA.

0

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9

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6

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0

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％(

)

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Figure3TherecoveryofDNAasafunctionoftheamountofDNAinsuspension.The

recoveryofDNAwasobservedtobedepend