Bisulfite Modification of DNA
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| Source:ProtocolOnline |
| Abstract:ModifyingDNAusingsodiumbisulfitetoconvertunmethylatedcytosinestouracilsandsubsequentlydetectmethylatedcytosinesusingmethylationspecificPCR(MSP)techniqueorbisulfitegenomicsequencingafterPCRamplificationwithorwithoutcloning.ThisprotocolalsodescribesprocedureforhandlingnanogramquantitiesofDNA. |
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MaterialsandRegents Prepareimmediatelybeforeuse - 10mMhydroquinone(Sigma;#H9003)
- 40.5%sodiumbisulfite(Sigma;#S8890,8.1g/20ml),dissolve8.1gsodiumbisulfitein17mlcoldwater,adjustpHto5.0with10NNaOH,addwatertobringvolume20ml)
Dissolveabovesolutionsbygentlyinvertingwithaminimumamountofmixing.KeepsolutionscoldandinthedarkasmuchaspossIBLe. Procedure - Digest2-5µgofhigh-molecular-weightgenomicDNAovernightwitharestrictionenzymethatdoesnotdigesttheDNAwithintheregionofinterest.
- ExtracttheDNAwithphenol:chloroform:isoamylalcohol(PCI;25:24:1),precipitatewith1/10volume5Mammoniumacetateand2volumesethanolat-85¡ãCfor15min.Centrifuge(14,000rev/min)atroomtemperature,removethesupernatantandwashthepellettwicewith70%ethanol.DrythepelletandresUSPendin100µlTE(10mMTris-HCl,pH7.5,1mMEDTA).
- DenaturetheDNAbyaddingfreshlypreparedNaOH(3M)toafinalconcentrationof0.3M.Incubateat42¡ãCfor30min.
- Toasiliconizedmicrocentrifugetubeadd:
- 1020µl40.5%sodiumbisulfite
- 60µl10mMhydroquinone
- 110µlDNA(+NaOH)
- 10µlwater
- Gentlymixandoverlaywithmineraloil.Coverthetubewithaluminumfoiltoshieldfromthelight.
- Incubateat55¡ãCfor16-18h.
- PurifyDNAusingtheGenecleanIIkit(IntermountainScientificCorporation)oranyothermethodsofyourchoice.
- Afterpurification,resuspendDNAandaddTEtoafinalvolumeof100µl.
- DenaturethesamplewithfreshlypreparedNaOH(asabove)andincubateat37¡ãCfor15min.
- Neutralizebyaddingammoniumacetate(pH7.0)to3M.
- PrecipitatetheDNAwiththreevolumesofethanol,centrifugefor10min(14,000rev/min)atroomtemperature,washtwicewith70%ethanolanddryunderavacuum.Resuspendin50µlTE,andstoreat-20¡ãCwrappedinfoil.TreatedDNAshouldbeusedwithinonemonthasdegradationmayoccurinthecleanedandfrozensample.
BisulfitemodificationfornanogramquantitiesofDNA FormodificationsofsmallerquantitiesofDNAsuchasDNAfrommicrodissectedtissues.Thebasicchangesthatismadetotheaboveprotocolincludethemethodofextractionandtheadditionofmusselglycogentoprecipitations. Thestepsarethesameasaboveandonlychangesarenoted. - InStep4,thebisulfitereactionisscaleddowntoaccommodatethesmallervolumeofDNA:
- 255µlsodiumbisulfate
- 15µlhydroquinone
- 27.5µlDNA(+NaOH)
- 2.5µlwater
- InStep8,TEisaddedtoafinalvolumeof25µl.
- InStep11,theprecipitationisperformedinthepresenceof40µgmusselglycogenat-80¡ãCfor30min,followedbycentrifugation(14,000rev/min)atroomtemperaturefor30min.TheDNAisresuspendedin25µlTE.
Note - ItisimportantthattheDNAiscompletelydenaturedpriortoandinthepresenceofthebisulfitesolutionorthemodificationwillnotbecomplete.
- Toensurecompletedenaturation,nomorethan5µg(orless)ofstartingmaterial(DNA)shouldbeused
- Theinitialalkalinedenaturationshouldbeat42¡ãCfor30min.
- TheDNAdigestionwithrestrictionenzymescanbeomitted.
Reference- Tremblay,KD.1998TechnicalTipsOnline.
- Clark,SJetal.NucleicAcidsRes1994;22:1827.
- Li,LCandDahiya,R.Bioinformatics,2002;18(11):1427.
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