foki是什么意思i在线翻译读音例句

Nearest-NeighborAnalysis9

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From:MethodsinMolecularBiology,vol.200:DNAMethylationProtocols

Editedby:oyeHumanaPressInc.,Totowa,NJ

2

Nearest-NeighborAnalysis

oye

uction

Nearest-neighboranalysiscanbeusedtoidentifythe3′nearestneighbors

of5mCresiduesinDNA(1,2).Itcanalsobeusedtomeasurethelevelof

methylationofaspecifilly,inthe

caseofmammalianDNA,thismeansquantifyingthedegreeofmethylation

headdedadvantageofbeingapplicabletosmall

ydrawbackis

thatitisaradioactivetechniqueandtheappropriatefacilitiesandtechniques

forhandlingradioactivesubstancesmustbeavailable.

eoftheProcedure

DNAisdigestedwitharestrictionenzymeandlabeledatarestrictionenzyme

cutsitewithKlenowfragmentofDNApolymeraseIanda[-32P]dNTP.

AfterdigestionofthelabeledDNAtodeoxyribonucleotide3′-monophosphates

usingacombinationofanexonucleaseandanendonuclease,theradiolabeled

5′-phosphateofthe[-32P]dNTPwillappearasthe3′-phosphateofthe

nucleo回眸一笑百媚生赏析 tide(X)thatwasimmediately5′itintheDNA(itsnearestneighbor).

Aslabelingistemplatedependent,theamountofthelabelednucleotide

3′-monophosphateinthedigestreflectsthefrequencyofadinucleotide(XpN)

hniqueoflabelingcutsitesbyfill-inreactionasdescribed

hereisamodificationofthenick-labelingnearest-neighboranalysistechnique

firstpublishedbyGruenbaumetal.(3).Intheauthor’sexperience,theoriginal

techniqueofusingDNaseItonicktheDNAandt安组词 heDNApolymeraseI

holoenzymetolabeltheDNAbynicktranslationgiveslessreproducibleresults

thanthefill-inmethodusingKlenowfragmentofDNApolymeraseI.

10Ramsahoye

ficationofCpGMethylationinMammalianDNA

IfMbo(GATC)isusedtocuttheDNAand[-32P]dGTPisusedtolabelit,

afterdigestionofthelabeledDNAtodeoxyribonucleotide3′-monophosphates,

thequantitiesoflabeled5mdCp,dCp,Tp,dGp,anddApreflecttherelative

frequenciesofthedinucleotides5mdCpG,dCpG,TpG,dGpG,anddApGat

MboIcutsitesintheDNA.

ficationofNon-CpGandCpGMethylation

Ifthesequencecontextofcytosine-5methylationisunknownitmaynotbe

thylationispresentinsequences

otherthanCpGtheDNAcanbecutwithFokI(GGATGN

9–13

)andlab忆江南的作者是谁? eled

separatelywitheachofthe4[-32P]ucleotidescontaininga

5′ionalfor

usingFokIhereisthatevenifthemethylationonlyoccurredwithinaspecific

sequenceintheDNA(a4–6baserecognitionsequence)therewouldbean

approx1in1000chancethatsuchsiteswouldalsohaveaFokIrecognition

methylationoccurred

consistentlywithina4–6basesequencecontextitshouldbedetectableusing

thistechnique,ldbenotedthatthereisahypothetical

possibilitythatmethylationcouldbemissedifthepatternofmethylationin

thesamplewassuchthatitalwaysarose9–13basesdownstreamofaspecific

okIinthisinstancemightpositivelyexcludethe

,ifthegenomeoftheorganismwas

particularlysmall(oftheorderof106bases)andmethylationoccurredwithin

aspecific5or6basesequenceonly,toofewmethylatedsitesmightbepresent

downstreamofaFokIsitetoreliablyallowtheirdetectionusingthisenzyme.

als

ts

lecular-weightDNA.

ictionenzymethatreliablecutscytosine-5methylatedDNAleavinga5

,FokIforthedetectionof5mCat5mCpN,MboIforthedetection

of5mCat5mCpGandMvaIfordetectingmethylationoftheinternalcytosine

inthesequenceCCWGG.

3.[-32P]dNTP(3000Ci/mmol,AmershamPharmaciaBiotech).

fragmentofDNApolymeraseI+labelingbuffer(AmershamPharmacia

Biotech).

occalnuclease(P6752,Sigma).

leenphosphodiesterase(WorthingtonBiochemicalCorporation).

Nearest-NeighborAnalysis11

occalnuclease/spleenphosphodiesterasedigestionbuffer:15mMCaCl

2

,

100mMTris-HCl.

8.0.2Methylenediaminetetraaceticacid(EDTA)(Sigma).

exG50spincolumns(availablefromRoche).

onA:66volumesisobutyricacid:18volwater:3vol30%ammonia

solution.

onB:80volumessaturatedammoniumsulphate:18vol1Maceticacid:

2volisopropanol.

ent

ctivitylaboratoryequippedwithprotectivescreens.

ableglovesshouldbewornatalltimes.

athsetat15C.

cksetat37C.

,SpeedVac).

-layerchromatography(TLC)developingtanks.

-backed20cm20cmcelluloseTLCplates.

8.X-rayfilm.

9.X-raycassettes.

per.

orimagerorscintillationcounter.

tionofPercentMethylationatCpG

hestandardmethods

canbeusedbuttheDNAshouldbehighmolecularweightandfreeofRNA.

RNAshouldberemovedbyenzymatichydrolysiswithRNaseAandRnaseT1

(together)followedbyrecoveryoftheDNAbyethanolprecipitation.

1gDNAwith10unitsofMboIat37Covernight.

-inactivatetheenzyme(70Cfor20min).

itatetheDNAinethanol,pelletbycentrifugation,andre-dissolvethe

DNAin10theDNAisre-dissolving,prepareanappropriate

numberofSephadexG50columnsinorderthatthesearereadyforuseon

completionofthelabelingstep.

3L[-32p]dGTP(30Ci),1.5L10Xlabelingbuffer,and0.5L

Klenowonice.

tefor15minat15C.

2L0.2MEDTAtoterminatethereaction.

llytransferthelabelingmixturetothetopofaSephadexG50spin

column.

fugeat1100gfor4mincollectingtheflowthroughina1.5mLpolypro-

pylenetube.

12Ramsahoye

nthelabeledDNAinaDNAspeedvac.

theDNAinavolumeof7L(5Lmicrococcalnucleasedigestionbu一场秋雨一场寒是哪首古诗 ffer,

1L[0.2units]micrococcalnucleaseand1L[2g]spleenphosphodiesterase.

Thedigestshouldbecompleteafter4hat37C.

dtoTLCorfreezethesampleat–20CuntilreadytoproceedtoTLC.

ationofSephadexG50Columns

exG50columnscanbepurchasedfromcommercialsuppliers(Roche).

Theycanalsobepreparedmorecheaplyinhouseusing1-mLsyringes,swollen

SephadexG50,andglasswool(toplugthesyringeandpreventescapeof

sephadexduringcentrifugation).

areyourowncolumns,rollasmallamountofglasswoolbetweena

glovedfingerandthumbandinsertitintoa1-mLsyringeusingthesyringe

untofglasswoolshouldbesuchthatitisjustsufficientto

covertheexitholeofthesyringeandpreventtheescapeofsephadexduring

centrifugation.

ephadexG50slurryintothebarrelofthesyringeandfilltothebrim.

Insertthesyringeintoa15-mLtube(Falcon).

fugeat1100gfor2mintocompacttheG50andexpelthebuffer.

oreG50slurryintothebarrel(fillingtothebrim)andcentrifugeagain.

ple

shouldbeappliedtothecenterofthecolumnanda1.5-mLpolypropylene

tubeshouldbeplacedinthe15-mLFalcontubetocollecttheeluteafter

centrifugation.

fugethesampleat1100gfor4mintoseparatethelabeledDNA(which

appearsintheelute)fromthefreenucleotides(whichareretainedinthe

column).

-LayerChromatography

uthor’sexperience,TLCdevelopingtanksdesignedtotakemorethantwo

platesinnearverticalpositionsgivesuboptimalseparationsinthisapplication.

Standardtanksthatallowforallowamaximumoftwoplatestobed适应的近义词 eveloped

atonce,giveimprovedseparationsastheplatescanbepositionedatamore

favourableangle(Fig.1A).

shouldbelabeledtoahighspecifirilythetube

containingthedigested32P-labeledDNAshouldreadmorethan2000counts/s

whenplacedupagainstaGeigercounter.

2-Lpipet,spot0.3Lofthedigestontoa2020cmglass-backed

renottomark

teshouldbelabeledwithapencilinthe

topleftcorner(Fig.1B).Theposition(forapplication)canbemarkedlightly

NAisinsufficientlylabeled(therewastoo

littleDNA)thenthesamplemayhavetobeappliedrepeatedly(withintervening

Nearest-NeighborAnalysis13

drying)ouldbeavoidedifpossibleasitwillinevitable

yasingle

0.3Lapplications关于中秋的古诗词句 houldgiveameasurementof500–1000counts/swhena

Geigercounterishelddirectlyoverit.

utionshouldbemadeupina

fumehood(isobutyricacidfumesarefoul-smellingandtoxic)andallsubsequent

chromatographyshouldbecarriedoutinthefumehood.

44mLofsolutionAintoaTLCdevelopingtankcompletewithglasslid.

Ensurethatthereisagoodseal.

eappliedsampleisdry,carefullyplacetheTLCplateatananglein

thedevelopingtankandreplacethelid(Fig.1A).Allowtheplatetodevelop

ouldtake12–18h,thetimetakenbeingdependentontheambient

tionsarequickerbutnoticeablypoorerinthesummer

evatedambienttemperatureisaproblemattemptsshouldbemade

tocarryoutthechromatographyinanair-conditionedroom.

eplateisfullydeveloped,removeitcarefullyandplaceitonabsorbent

paper(cellulosesideuppermost)behindaradiationscreenwiththefume-hood

lete

dryingoftheplateadverselyaffectsthesubsequentchromatography.

Fig.1.(A)TLCdevelopingtanks.(B)ApplyingthesampletotheTLCplate.

14Ramsahoye

utionAinthedevelopingtankshouldthenbepouredoffintoacontainer

forsolventwasteandthetankshouldthenbewashedoutthoroughlyinwater

(takingcarenotthesplashthedryingTLCplate).

eferableforallofthesestepstobecarriedoutinthefumehood(if

equippedwithasink)astheresidualisobutyricacidwillleaveafoulsmelleven

inawell-ventilatedroom.

eTLCplateisdry,turntheplatethrough90degreesandsubjectthe

sampletotheseconddimensionofchromatographyusingsolutionB.

eseconddimensioniscomplete,removetheplateanddryagaininthe

withtheextractoronisessentialas

otherwisecoarsecrystallizationoftheammoniumsulphateleadstodeterioration

andflakingofthecelluloselayer.

yingiscompletetheplatescanbeana体现汉字特点的对联 lyzedbyautoradiographyor

aseofautoradiographythelabelednucleotidescan

subsequentlybequantifissibletofitfourTLCplates

inone3543cmautoradiog闩怎么读 raphycassetteiftheyarestackedasindicatedin

vesonX-rayfilmandsoismoreeconomical.A24-hexposureis

usuallysufficienttolocateevenlowlevelsofmethylation.

evelopingthefilm,theautoradiographisusedtolocatethepositionofthe

labelednucleotidesontheTLCplates(Fig.3).Tracingpaperisusedtorecord

thepositionswithapencil,cing

papercanthenbeapplieddirectlytotheplateandapencilusedtodelineatethe

positionsoftherespectivenucleotidesontheplate.

ementforstackingfourTLCplatesinasingleautoradiography

cassette.

Nearest-NeighborAnalysis15

lulosecanthenbescrapedoffintoscintillationvialsusingcleanscalpel

blades(beingcarefultorecoverallofthelabelandnottocross-contaminate

othernucleotides).

ioactivityinthecellulosecanthenbemeasuredbyscintillationafterthe

teandreproduciblequantificationsareonlypossible

ifgreatcareistakentoensurethatallthecelluloseisrecoveredwithoutspillage.

untingtherelativelyhigh-energyemissionsfrom32P,itisusually

sufficienttorecordthescintillationdatainunitsofc不知秋思落谁家上一句是什么 ountsperminute(CPM).

Thisisbecause32Pemissionsarenotsignificantlyquenchedinthecircumstances

uctionofaquenchcurveandconversionofthedatato

disintegrationsperminute(DPM)ewouldnot

applyifusingtheweakeremitting33Pisotopeinlabelingreactions.

References

,F.,Ramsahoye,B.H.,andJaenisch,R.(2000)DNAmethylationin

408,538–540.

oye,B.H.,Biniszkiewicz,D.,Lyko,F.,Clark,V.,Bird,A.P.,andJaenisch,R.

(2000)Non-CpGmethylationisprevalentinembryonicstemcellsandmaybemedi-

97,5237–5242.

aum,Y.,Stein,R.,Cedar,H.,andRazin,A.(1981)MethylationofCpG

tt.124,67–71.

edpositionsofnucleotide3′-monophosphatesaftertwodimensions

ofchromatography.

16Ramsahoye

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