nucleic acid extraction methods: dna - city university of...
TRANSCRIPT
-
MolecularDiagnostics Fundamentals,MethodsandClinicalApplicationsSecondEdition
Copyright 2012 F.A. Davis Company
NucleicAcidExtractionMethods:DNA
Chapter4
-
MolecularDiagnostics Fundamentals,MethodsandClinicalApplicationsSecondEdition
Copyright 2012 F.A. Davis Company
Objectives Compareandcontrastorganic,inorganic,andsolidphase
approachesforDNAisolation. Compareandcontrastorganicandsolidphaseapproachesfor
isolatingtotalRNA. DistinguishbetweentheisolationoftotalRNAwiththatof
messengerRNA. Describethegelbased,spectrophotometric,andfluorometric
methodsusedtodeterminethequantityandqualityofDNAandRNApreparations.
CalculatetheconcentrationandyieldofDNAandRNAfromagivennucleicacidpreparation.
-
MolecularDiagnostics Fundamentals,MethodsandClinicalApplicationsSecondEdition
Copyright 2012 F.A. Davis Company
Specimens Blood Buffycoat Bonemarrow Solidtissue Lavagefluids Bacteria,viruses Fungi Organelles,mitochondria
-
MolecularDiagnostics Fundamentals,MethodsandClinicalApplicationsSecondEdition
Copyright 2012 F.A. Davis Company
SpecimenPreparation Bonemarrow,peripheralblood Densitygradientcentrifugation Differentialosmolysis
Tissue Freeze/crush Mince Enzymaticdigestion proteinaseKdigestion
Plants/fungi Homogenize Vortexwithglassbeads
-
MolecularDiagnostics Fundamentals,MethodsandClinicalApplicationsSecondEdition
Copyright 2012 F.A. Davis Company
IsolationofDNA PreparingtheSample
NucleatedCellsinSuspension(BloodandBoneMarrowAspirates) Fordifferentialdensitygradientcentrifugation,wholebloodorbonemarrow
mixedwithisotonicsalineisoverlaidwithFicoll.Uponcentrifugation,themononuclearWBCs(thedesiredcellsforisolationofnucleicacid)settleintoalayerintheFicoll gradientthatisbelowthelessdenseplasmacomponentsandabovethepolymorphonuclear cellsandRBCs.
Fordifferentiallysis (differentialosmoticfragility),Incubationofwholebloodorbonemarrowinhypotonic bufferorwaterwillresultinthelysis oftheRBCsbeforetheWBCs.TheWBCsarethenpelletedbycentrifugation,leavingtheemptyRBCmembranes(ghosts)andhemoglobin,respectively,insuspensionandsolution.
-
MolecularDiagnostics Fundamentals,MethodsandClinicalApplicationsSecondEdition
Copyright 2012 F.A. Davis Company
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
-
MolecularDiagnostics Fundamentals,MethodsandClinicalApplicationsSecondEdition
Copyright 2012 F.A. Davis Company
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
-
MolecularDiagnostics Fundamentals,MethodsandClinicalApplicationsSecondEdition
Copyright 2012 F.A. Davis Company
SpecimensAdequateforamplificationbypolymerasechainreaction(PCR)
Driedblood Saliva Bone,teeth Amnioticfluid Hairfollicles,hairshafts Buccalcells Cerebrospinalfluid Fixedtissue Feces Soil
-
MolecularDiagnostics Fundamentals,MethodsandClinicalApplicationsSecondEdition
Copyright 2012 F.A. Davis Company
DNAExtractionMethods
Organic:usesorganicchemicals,phenol,chloroform
Inorganic:usesinorganicchemicals,detergents,ethylenediamine tetraacetic acid(EDTA),aceticacid,salt(saltingout,spooling)
Solidphase:DNAisimmobilizedonasolidsupport,beads,orcolumns
-
MolecularDiagnostics Fundamentals,MethodsandClinicalApplicationsSecondEdition
Copyright 2012 F.A. Davis Company
OrganicDNAIsolation
-
MolecularDiagnostics Fundamentals,MethodsandClinicalApplicationsSecondEdition
Copyright 2012 F.A. Davis Company
InorganicDNAIsolation
-
MolecularDiagnostics Fundamentals,MethodsandClinicalApplicationsSecondEdition
Copyright 2012 F.A. Davis Company
SolidPhaseDNAIsolation Solidphase
isolationmediaincludesilicaspincolumns andbeads.
Nucleicacidbindingtosilicabeadsisthebasisformanyautomatedextractionsystems.
-
MolecularDiagnostics Fundamentals,MethodsandClinicalApplicationsSecondEdition
Copyright 2012 F.A. Davis Company
OtherDNAExtractionMethods
Limitingspecimens(fixedtissue,driedblood,bone) Rapidextractionforroutinetesting
-
MolecularDiagnostics Fundamentals,MethodsandClinicalApplicationsSecondEdition
Copyright 2012 F.A. Davis Company
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
-
MolecularDiagnostics Fundamentals,MethodsandClinicalApplicationsSecondEdition
Copyright 2012 F.A. Davis Company
DNAExtractionfromFixedTissueforPCR
-
MolecularDiagnostics Fundamentals,MethodsandClinicalApplicationsSecondEdition
Copyright 2012 F.A. Davis Company
MitochondrialDNA
Isolatemitochondriabycentrifugation. Slowcentrifugation Fastcentrifugation
IsolatetotalDNA.
-
MolecularDiagnostics Fundamentals,MethodsandClinicalApplicationsSecondEdition
Copyright 2012 F.A. Davis Company
MethodstoAssessDNA Gelelectrophoresiswith
knownstandards
Spectrophotometry1OD260 =50g/mLdsDNA(concentration)g/mLxmL=gDNA(yield)OD260 /OD280~1.62.0(purity)
Genomic DNA should look like this:
-
MolecularDiagnostics Fundamentals,MethodsandClinicalApplicationsSecondEdition
Copyright 2012 F.A. Davis Company
NucleicAcidExtractionMethods:RNA
Chapter4
-
MolecularDiagnostics Fundamentals,MethodsandClinicalApplicationsSecondEdition
Copyright 2012 F.A. Davis Company
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)
-
MolecularDiagnostics Fundamentals,MethodsandClinicalApplicationsSecondEdition
Copyright 2012 F.A. Davis Company
SpecimenCollection Bonemarrow,peripheralblood Acidcitratedextrose(ACD) LiquidK3EDTA (Heparin) RNAtubes
Tissue Freshinsaline:processimmediately Frozen, 70C,nitrogen Fixed,embedded
-
MolecularDiagnostics Fundamentals,MethodsandClinicalApplicationsSecondEdition
Copyright 2012 F.A. Davis Company
SpecimenPreparation Bonemarrow,peripheralblood Densitygradientcentrifugation Differentialosmolysis(removeRBCornuclei)
Tissue Freeze/grind Crushindenaturant
Bacteria/fungi/plants Homogenize Vortexwithglassbeads Enzymaticdigestion(Zymolyase/lysozyme)
-
MolecularDiagnostics Fundamentals,MethodsandClinicalApplicationsSecondEdition
Copyright 2012 F.A. Davis Company
RNAIsolation
Organic Solidphase
-
MolecularDiagnostics Fundamentals,MethodsandClinicalApplicationsSecondEdition
Copyright 2012 F.A. Davis Company
OrganicRNAIsolation GITC:strongRNAsedenaturant
-
MolecularDiagnostics Fundamentals,MethodsandClinicalApplicationsSecondEdition
Copyright 2012 F.A. Davis Company
SolidPhaseRNAIsolation
-
MolecularDiagnostics Fundamentals,MethodsandClinicalApplicationsSecondEdition
Copyright 2012 F.A. Davis Company
Macrodissect ormicrodissect
Digest with proteinase K
Organic extractionand precipitation
~20 micronsections
RNAExtractionfromFixedTissue
-
MolecularDiagnostics Fundamentals,MethodsandClinicalApplicationsSecondEdition
Copyright 2012 F.A. Davis Company
TypesofRNA MessengerRNA (mRNA) RibosomalRNA(rRNA) TransferRNA(tRNA) HeteronuclearRNA(hnRNA) SmallnuclearRNA(snRNA) DoublestrandedRNA(dsRNA) Manysmall/microRNAs
-
MolecularDiagnostics Fundamentals,MethodsandClinicalApplicationsSecondEdition
Copyright 2012 F.A. Davis Company
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
-
MolecularDiagnostics Fundamentals,MethodsandClinicalApplicationsSecondEdition
Copyright 2012 F.A. Davis Company
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
-
MolecularDiagnostics Fundamentals,MethodsandClinicalApplicationsSecondEdition
Copyright 2012 F.A. Davis Company
CommonContaminantsandTheirWavelengthsofPeakAbsorbance Wavelength(nm) Contaminant
230 Organiccompounds 270 Phenol 280 Protein >330 Particulatematter
-
MolecularDiagnostics Fundamentals,MethodsandClinicalApplicationsSecondEdition
Copyright 2012 F.A. Davis Company
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.
-
MolecularDiagnostics Fundamentals,MethodsandClinicalApplicationsSecondEdition
Copyright 2012 F.A. Davis Company
Summary DNAisextractedbyorganic,inorganic,andsolidphase
methods. DNAcanalsobeextractedbymorerapidmethodsormethods
designedforchallengingspecimens. RNAextractionmethodsincludeorganicandsolidphase
methods. mRNAcanbespecificallyextractedusingimmobilizedpolyTor
polyU. DNAandRNAconcentration,yield,andpurityareassessed
usinggelanalysis,spectrophotometry,orfluorometry.