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AnalysisofmethylationusingbisulphitesequencingContributedbyDr.A.Gratchev

Thismethodallowspreciseanalysisofmethylationinacertainregionbyconvertingallnonmethylatedcytosinesintotymines,whilemethylatedcytosinesremainunchanged.ThismethodrequiressmallamountofgenomicDNAandthereforeseemstobeveryusefulfortheanalysisofclinicalsamples,wherethematerialamountislimited.HoweverIsuggestoptimisingthemethodusinggenomicDNAfromacelllineandthenapplyittovaluablesamples.

BeforestartingtheexperimentyouhavetodevelopprimersforbisulphiteconvertedDNA.YoucangenerateamodelofbisulphitetreatedDNAbysubstitutingallcytosineswhicharenotinCGcontextintotymines.AndthendesignyourprimersinthewaythattheydonnotcontainanyCG.IfthisisimpossIBLe,youhavetouseC/TattheplaceofCinCGcontext.Usuallyprimerselectionisthemostcriticalinbisulphitebasedmethylationanalysis,sincethecomplexityofDNAisreduced.ThereforeIwouldsuggesttoselect2-3pairsofprimers,checkthemonbisulphitemodifiedDNA,andusethemostspecificones.

Protocol

IsolategenomicDNAwiththequalitysufficientforrestrictionenzymedigestion.
DigestDNA(50to200ng)withanyenzymewhichdoesnotcuttheregionofinterest,butresultinginasshortfragmentsaspossibleinsmallestpossiblevolume.Note:increasingtheamountofDNAwillmakedenaturingnotefficientenoughandthereforemakebisulphitereactionincomplete!
StopthereactionbyboilingDNAfor5min.
Add10NNaOHto0.3NfinalconcentrationanddenatureDNA37°Cfor15min.
Prepare4-5Eppendorftubeswithcoldmineraloil.
Add2xvolumeof2%lowmeltingagarosetotheDNAsolution,mixbypipettingupanddown.
Formagarosebeadsbypipetting10µlaliquotsofDNA/agarosemixtureintocoldmineraloil.Note:Don"tpipettthesecondaliquotinthetubewhereyoualreadyhaveonebead!
Transferbeadsinthetubecontaining1mlofmodifyingsolution(5Msodiumbisulphite(2.5MsodiummetABIsulphite),100mMHydroquinon).
Incubatethetubes4hat50°Cinthedark.
Washthebeads6timesfor15mininTEpH8.0.
Completethemodificationbyincubatingthebeads2timesfor15minin0.2NNaOH.
Washthebeads3timesfor15minindoubledistilledH₂O.
UseoneuloftheobtainedDNAforPCRwithselectedprimers.

ObtainedPCRproductcanbesequenceddirectly,inthiscaseyouwillobtainmoreorlessreliableresultsaboutthepercentageofmethylatedcytosineineveryposition.Anotherpossibilityistoclonetheproductandthensequence10ormoreindividualclones.Thismethodismuchmoretimeconsuming.ThirdmethodwhichcanbeusedfortheanalysisofbisulphitetreatedDNAisSingleNucleotidePrimerExtension(SNuPE).

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