Nucleic Acid Extraction Methods: DNA - City University of...

MolecularDiagnostics Fundamentals,MethodsandClinicalApplicationsSecondEdition

Copyright 2012 F.A. Davis Company

NucleicAcidExtractionMethods:DNA

Chapter4



Objectives Compareandcontrastorganic,inorganic,andsolidphase

approachesforDNAisolation. Compareandcontrastorganicandsolidphaseapproachesfor

isolatingtotalRNA. DistinguishbetweentheisolationoftotalRNAwiththatof

messengerRNA. Describethegelbased,spectrophotometric,andfluorometric

methodsusedtodeterminethequantityandqualityofDNAandRNApreparations.

CalculatetheconcentrationandyieldofDNAandRNAfromagivennucleicacidpreparation.



Specimens Blood Buffycoat Bonemarrow Solidtissue Lavagefluids Bacteria,viruses Fungi Organelles,mitochondria



SpecimenPreparation Bonemarrow,peripheralblood Densitygradientcentrifugation Differentialosmolysis

Tissue Freeze/crush Mince Enzymaticdigestion proteinaseKdigestion

Plants/fungi Homogenize Vortexwithglassbeads



IsolationofDNA PreparingtheSample

NucleatedCellsinSuspension(BloodandBoneMarrowAspirates) Fordifferentialdensitygradientcentrifugation,wholebloodorbonemarrow

mixedwithisotonicsalineisoverlaidwithFicoll.Uponcentrifugation,themononuclearWBCs(thedesiredcellsforisolationofnucleicacid)settleintoalayerintheFicoll gradientthatisbelowthelessdenseplasmacomponentsandabovethepolymorphonuclear cellsandRBCs.

Fordifferentiallysis (differentialosmoticfragility),Incubationofwholebloodorbonemarrowinhypotonic bufferorwaterwillresultinthelysis oftheRBCsbeforetheWBCs.TheWBCsarethenpelletedbycentrifugation,leavingtheemptyRBCmembranes(ghosts)andhemoglobin,respectively,insuspensionandsolution.



IsolationofDNA PreparingtheSample

TissueSamples Grindingthefrozentissueinliquidnitrogen,homogenizingthetissue,orsimplymincingthetissueusinga

scalpelcandisruptwholetissuesamples. Fixed,embeddedtissuemaybedeparaffinized bysoakinginxylene (amixtureofthreeisomersof

dimethylbenzene).Lesstoxicxylenesubstitutes,suchasHistosolve,Anatech ProPar,orParaClear,arealsooftenusedforthispurpose.Afterxylenetreatment,thetissueisusuallyrehydratedbysoakingitindecreasingconcentrationsofethanol.Alternatively,fixedtissuemaybeuseddirectlywithoutdewaxing.

TissueFixativesInfluencingNucleicAcidQualityRelativeQuality AverageFragment

Fixative ofNucleicAcid SizeRange(kb) 10%buffered Good 2.05.0 neutralformalin Acetone Good 2.05.0 Zambonis Notasgood 0.22.0 Clarkes Notasgood 0.81.0 Paraformaldehyde Notasgood 0.25.0 Metharcan Notasgood 0.71.5 Formalinalcohol Notasgood 1.04.0 aceticacid B5 lessdesirable



IsolationofDNA PreparingtheSample Microorganisms Enzymes lyzozyme orzymolyase digestcellwallpolymers

Treatmentwithdetergent(1%sodiumdodecylsulfate)andstrongbase(0.2MNaOH)inthepresenceofTris base,ethylenediaminetetraacetic acid(EDTA),andglucosecanbreakbacterialcellwalls.

Boilingindilutesucrose,TritonX100detergent,Tris buffer,andEDTAafterlysozymetreatmentreleasesDNA thatcanbeimmediatelyprecipitatedwithalcohol

Mechanicalforce grindingormixingwithglassbeads



SpecimensAdequateforamplificationbypolymerasechainreaction(PCR)

Driedblood Saliva Bone,teeth Amnioticfluid Hairfollicles,hairshafts Buccalcells Cerebrospinalfluid Fixedtissue Feces Soil



DNAExtractionMethods

Organic:usesorganicchemicals,phenol,chloroform

Inorganic:usesinorganicchemicals,detergents,ethylenediamine tetraacetic acid(EDTA),aceticacid,salt(saltingout,spooling)

Solidphase:DNAisimmobilizedonasolidsupport,beads,orcolumns



OrganicDNAIsolation



InorganicDNAIsolation



SolidPhaseDNAIsolation Solidphase

isolationmediaincludesilicaspincolumns andbeads.

Nucleicacidbindingtosilicabeadsisthebasisformanyautomatedextractionsystems.



OtherDNAExtractionMethods

Limitingspecimens(fixedtissue,driedblood,bone) Rapidextractionforroutinetesting



Chelex removesmultivalent cations.

Heat tissue (hair roots, saliva, etc.) in 300 L 5%20% Chelex 100 resin [cation chelating resin].

DNA is insupernatant.

DNAPurificationwithChelex ResinforPCR



DNAExtractionfromFixedTissueforPCR



MitochondrialDNA

Isolatemitochondriabycentrifugation. Slowcentrifugation Fastcentrifugation

IsolatetotalDNA.



MethodstoAssessDNA Gelelectrophoresiswith

knownstandards

Spectrophotometry1OD260 =50g/mLdsDNA(concentration)g/mLxmL=gDNA(yield)OD260 /OD280~1.62.0(purity)

Genomic DNA should look like this:



NucleicAcidExtractionMethods:RNA

Chapter4



LaboratoryPreparation Bench,equipment

SeparatelaboratoryareadesignatedRNaseFree(RNF). WipewithRNaseZAP,RNaseAWAY.

Disposables CertifiedRNasefree Rinsedin0.1%diethylpyrocarbonate(DEPC)

Reagents CertifiedRNasefree Add0.05%0.1%DEPC(exceptTris) TestwithRNaseAlert(Ambion,Inc.)

Reactions AddRNasin(Promega)



SpecimenCollection Bonemarrow,peripheralblood Acidcitratedextrose(ACD) LiquidK3EDTA (Heparin) RNAtubes

Tissue Freshinsaline:processimmediately Frozen, 70C,nitrogen Fixed,embedded



SpecimenPreparation Bonemarrow,peripheralblood Densitygradientcentrifugation Differentialosmolysis(removeRBCornuclei)

Tissue Freeze/grind Crushindenaturant

Bacteria/fungi/plants Homogenize Vortexwithglassbeads Enzymaticdigestion(Zymolyase/lysozyme)



RNAIsolation

Organic Solidphase



OrganicRNAIsolation GITC:strongRNAsedenaturant



SolidPhaseRNAIsolation



Macrodissect ormicrodissect

Digest with proteinase K

Organic extractionand precipitation

~20 micronsections

RNAExtractionfromFixedTissue



TypesofRNA MessengerRNA (mRNA) RibosomalRNA(rRNA) TransferRNA(tRNA) HeteronuclearRNA(hnRNA) SmallnuclearRNA(snRNA) DoublestrandedRNA(dsRNA) Manysmall/microRNAs



MessengerRNA(mRNA)

TheefficiencyofpolyA andpolyUbindingisvariable.

Secondarystructureinthetargetsamplemaycompetewithbindingtothecaptureoligomer.

mRNAswithshortpolyA tailsmaynotbindefficientlyoratall.

ATrichDNAfragmentsmightbindtothecolumnandnotonlycompetewiththedesiredmRNAtargetbutalsocontaminatethefinaleluant.

A A A A A A A A A

T T T T T T T T T

Bead or column



MethodstoAssessRNA Gelelectrophoresis(totalRNA)(quality)

Spectrophotometry1OD260 =40g/mLRNA(concentration)g/mLxmL=gRNA(yield)OD260/OD280 >1.6(purity)

FluorometryRiboGreen

Total RNA should look like this.

--28S rRNA

--18S rRNA

M

M = molecular weight marker



CommonContaminantsandTheirWavelengthsofPeakAbsorbance Wavelength(nm) Contaminant

230 Organiccompounds 270 Phenol 280 Protein >330 Particulatematter



Fluorometry DNAspecificdyeHoechst33258{2[2(4hydroxyphenyl)(6benzimidazol)]6(1

methyl4piperazyl)benzimidazol/.3HCl}.ThisdyecombineswithadeninethyminebasepairsintheminorgrooveoftheDNAdoublehelixandisthusspecificforintactdoublestrandedDNA.

PicoGreen andOliGreen (MolecularProbes,Inc.)areotherDNAspecificdyesthatcanbeusedforfluorometric quantification.DuetobrighterfluorescenceuponbindingtodoublestrandedDNA,PicoGreen ismoresensitivethanHoechstdye.

OliGreen isdesignedtobindtoshortpiecesofsinglestrandedDNA(oligonucleotides).OliGreen willnotfluorescewhenboundtodoublestrandedDNAorRNA.

Fluorometry measurementsrequirecalibrationoftheinstrumentwithaknownamountofstandardbeforemeasurementofthesample.



Summary DNAisextractedbyorganic,inorganic,andsolidphase

methods. DNAcanalsobeextractedbymorerapidmethodsormethods

designedforchallengingspecimens. RNAextractionmethodsincludeorganicandsolidphase

methods. mRNAcanbespecificallyextractedusingimmobilizedpolyTor

polyU. DNAandRNAconcentration,yield,andpurityareassessed

usinggelanalysis,spectrophotometry,orfluorometry.

Nucleic Acid Extraction Methods: DNA - City University of...

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