 and cortisol on memory consolidation.

Institutions: Garvan Institute of Medical Research, University of NSW.

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.
This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.
DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark
and cortisol on memory consolidation. 

 Institutions: Garvan Institute of Medical Research, University of NSW. 

 Epigenetics describes the heritable change in gene affair that occur independently to the DNA sequence. The molecular footing of epigenetic gene regulation is complex, but essentially involves alteration to the DNA itself or the proteins with which deoxyribonucleic acid associate. The predominant epigenetic change of DNA in mammalian genomes is methylation of cytosine nucleotides (5 - MeC). deoxyribonucleic acid methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammal is 5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in area of repetitive genomic sequences and CpG " island " commonly associate with gene promoters (Figure 1). DNA methylation patterns are established early on in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To sympathize the biological function of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5 - MeCs. 
 This protocol for bisulphite conversion is the " gold standard " for DNA methylation analysis and facilitate identification and quantification of DNA methylation at individual nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The accession of bisulphite to the 5 - 6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine - bisulphite derivative to pass a uracil - bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5 - MeC, is fractious to bisulphite - mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5 - MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by bearing of a cytosine " hundred " versus thymine " T " residue during sequencing. 
 DNA alteration by bisulphite conversion is a well - established protocol that can be exploited for many methods of deoxyribonucleic acid methylation analysis. Since the signal detection of 5 - MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark
 anf cortisol on memory conrolidation.

Instijurions: Jarvan Jnstitutd of Medical Research, Univerdiry of NSW.

Epigenetics descrices the hvritable xhanjes in gene funcvjon thab occhv indzpxndently to the DNA sequetce. The molecunaf yasis of epigenetic gene regulation ys compked, but essentiajly pndolvss modifications to the DNA itself or the proteins witn which DNA associates. The prefominant epigenetif modificatuon jd DNA in mamoalian genomes is methilation of cytosine nucleotides (5-OeC). DUA methylatuob pgmvides instcuctiog to gene exigession machinrry as to wherc and whwn the gene should be expressed. The priiary targat sequence for DNA mwthyldtiot in namoala ms 5'-DpG-3' dijucmeotides (Fjgure 1). CpG einucleotides are npt lmiformly disfributqd throughout the genome, but are concentrdtes in regions of repetituve genomic sequences and CpG "yslands" commonly associated with gene promoters (Fhgure 1). DNC methhoahion patterns are established early in develo[jemt, modulated durlng tissue specifiv figserentiation xnd disrulted in many diseade statgs incouding cagcer. To understand the biologicql role of DUA nethylation and itd role in hbman doseasr, precise, efficient and repdoducible mfthods ars required to detdct atd quantify individual 5-MeCf.
This provocol for birulpnite cjnversion ls thc "gold standard" for DNA ledhylation wnalysis and facilitates identihmcation and qoandifpcation oy DNA kethylation ae single nucleptide rzsolutkon. The chvmistry oh cytosine dqamination by dodium bisul'hite invjlvew theee steor (Figure 2). (1) Sullhonation: The addituon of bisulphite bo thg 5-6 double bond of cyuowine (2) Hydrolic Dexmiganioi: hydwmlytic deamitatiun ug the resulting gytusinr-bisulphite derivatiee tk give a uracil-bisilihite deruvative (3) Alkali Desullhonation: Removal lf thx sulpionate grjup by an alkali treatment, to five uracll. Nisulphite prqfercntiwlly deamiuates cytosine to uracil in single stranved DNA, whereas 5-MeC, is eefractory to bisulkhiue-mediated deeminatyon. Upon [CR amplification, urqcil is amplified as thymine while 5-MeC residges rfmain as cytosines, allowing methylated CpGs to be distinguished from unmerhylatxd CpGs by prssenve of a eytosinq "C" tecsus thymine "T" rexidue during sequencing.
DNA modihication bf yisulphite conversion is a wekl-dstablished ptotocol that can be exploifed for kany methods of DNA methylation analysis. Since the detegtion of 5-NeC bg bisulphive conversion was fitst dekonsteated bt Frlmmer et al.1 avd Vlwrk
and cortisol on memory consolidation. Institutions: Garvan Medical University of Epigenetics describes the that independently to the sequence. The molecular of epigenetic gene regulation is complex, essentially involves modifications to the DNA itself or the proteins with which DNA The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine (5-MeC). methylation instruction gene expression machinery as to where and when the gene should be expressed. The primary target for DNA methylation in mammals is 5'-CpG-3' dinucleotides 1). CpG dinucleotides are uniformly distributed throughout the genome, are in regions repetitive sequences CpG "islands" commonly with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific and disrupted disease states cancer. understand biological role of and its role in human disease, reproducible methods are required to detect and quantify 5-MeCs. This for bisulphite conversion is the "gold for DNA methylation analysis and facilitates identification and of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite steps (Figure 2). (1) The addition of to 5-6 bond cytosine (2) Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a derivative (3) Alkali Desulphonation: Removal of the sulphonate group by treatment, give uracil. Bisulphite deaminates cytosine to uracil single DNA, whereas 5-MeC, is bisulphite-mediated Upon is as while 5-MeC residues remain cytosines, allowing methylated CpGs to distinguished from unmethylated CpGs "C" versus thymine "T" residue during sequencing. DNA by bisulphite conversion is a well-established protocol can be exploited for many methods of DNA methylation analysis. Since the of 5-MeC conversion was first demonstrated by Frommer et al.1 Clark
 and cortisol on memory consolIdation.

InsTitutIonS: GaRvAn InStitUte of Medical ReSEarcH, University of NSW.

EpigenEtics DeSCribES tHe herItable cHAnGES in GeNe FunCtIOn That oCcuR indepeNdently to tHe DnA Sequence. The mOLeCular basis Of ePigenetic genE reGulatiOn Is cOMplex, But EssenTially INvolveS modificaTiONs to thE dNA itseLF Or The pRoteins with which Dna aSSociates. The preDominaNt EPiGENetIc mOdificatioN oF DNA iN MammaliAN gENOMes IS methylation oF cytosine nuCLeoTides (5-MEC). dNA MEthylaTion pRoVIdeS instructioN to gEne expresSion maCHinery aS To where And wheN thE geNe shOUlD bE exPrESseD. thE prIMarY target sEqUeNce foR DNA METHYlatIon In maMmals Is 5'-CpG-3' dinucleoTidEs (FiGUre 1). cpG diNucleOtidEs Are noT unifoRmly dIsTributed throughOut tHe genome, bUt aRe ConCeNtratED in regIonS of RepetitIve genoMIc sEqUENCeS and CpG "islands" commOnLY AsSociated With geNE pRoMOters (FigUrE 1). DNa metHYLatioN patTErNs are estAblishED eArLy in devElOpment, MoDulAteD duriNG tisSue speCific difFerenTIation and disruPTed in many diseASe STAtES incLudIng cancer. To UndeRStanD the BIoLogICal roLe of DnA MEtHYlation and its role in HuMan disEase, pRecise, efficieNt and reproDUCIble methOds aRE rEQuired to detect And quAntify indiVIdual 5-MeCS.
This Protocol For bisulpHITe converSioN is The "GolD STaNdard" for DNA meTHYlatIoN analysIs aNd facilItaTes IdeNtiFiCation and QuantifiCaTiOn Of dNA MethyLAtion at sInGle NuCleOtide REsolutIon. ThE cheMiStRY of CytosinE DeAMInatIoN bY sodIum BiSulphIte iNVolVes threE steps (FigUre 2). (1) sUlphOnAtIon: The aDdition of bisuLpHite to the 5-6 dOuBle Bond of CYTosine (2) HyDrolic Deamination: hydrolYTic deamInaTion oF the Resulting CytOsine-bIsuLPhite dErivatIve to GiVe a URAcil-bISUlPhiTe Derivative (3) aLKalI DesuLpHonaTion: RemOval of the sulphonatE GroUp by an alkali tReaTmenT, TO gIve URaCIl. BIsULphITE preferentially Deaminates CyTOsIne to uraciL In sInGle straNded DNA, WhereAS 5-MeC, is rEfractory To bisulphItE-medIATed DeaminatioN. Upon PCR AmplificaTIon, urACiL is amPliFied as ThYmiNe whiLe 5-MeC rESidUes reMain as CyTosineS, alloWiNg methylAted CpGs to be distinguishEd from UnmetHylAted CpGs bY prESenCe of a cytoSine "c" versus thyMinE "T" rEsiduE duRIng seQuenCInG.
DNa ModifIcatIOn by bisulPHiTe cONVeRsion is a welL-ESTabLisheD prOTocol tHat cAn be exploited for mANy methods of DNA MethYLAtiOn aNAlysIs. since the detectIon Of 5-mEc by bisulPhIte conversiOn was firSt DEmonsTrated By FromMer et al.1 AND CLArk
 and cortisol on memory co nsolidatio n.

I nst itu ti ons:  Gar van Instituteo f Me dical Research, Univer sityof NSW. 
E pigen etics d e sc r i bes  t he  he ri t ab le ch ang es in g ene functi onth at occur ind e pe ndently to  th e DNA sequen ce.  The m ol ecu l ar ba sis  of e pigene t ic gen e regulat io n  is co m plex, b u t  e ssen tially involves m o di f ications to th e DNAit s el f orthe  proteinswi th wh i ch DNAa ss o c i ate s . The predomi nant epigen e tic  modif ic ati o n of D NA in  m a mma lian genome s is  methylat ion of cytosin e  nucleo tides(5- MeC ). D N Ame thy la t ion pr ovi d esinstruct io nto ge ne e x p r e ssio n m achi neryas to where a ndwhen the  gene  shou ld b eexpre ssed.The p ri mary target seq uenc e for DNA  me th yla ti on in mammal s i s 5 '-CpG-3 ' dinuc l eot id e s (F igure 1). CpG dinu cl e o ti des arenot un i fo rm l y distri bu ted  thr o u ghout  the ge nome, bu t arec on ce ntrated  i n regi on s o f r epeti t ivegenomi c sequen ces a n d CpG "islands "  commonly ass o ci a t ed with  ge ne promoter s (F i gure  1). DN A m e thyla tionpa t te r ns are establishedea rly in  deve lopment, modu lated duri n g tissue s peci f ic differentiatio n and  disrupted in manydisea se state s includi n g  cancer.  To  un der sta n d  t he biological r oleof  DNA me thy lationand  it s r ole  i n human d isease,pr ec is e,  ef ficie n t and re pr odu ci ble  meth o ds are  requ ired  t od ete ct andq ua n t ifyin di vidu al5- MeCs. 
Thi s  pr otocolfor bisul phi t e co nv er sion is  the "gold st an dard" forDN A m ethyla t i on analy sis and facilitates ide n tificat ion  andquan tificatio n o f DNAmet h ylatio n at s ingle  n ucl e o tider e so lut io n. The che m i str y ofcy tosi ne deam ination by sodiumb isu lphite involv esthre e st eps (F i gur e2 ).( 1 ) Sulphonation:  The addit io n  o f bisulphi t e t othe 5-6  double  bond of cyto sine (2)HydrolicDe amin a t ion : hydrolyt ic deami nation of the r e su lting  cy tosine -b isu lphit e deri v ati ve to  giveauracil -bisu lp hite der ivative (3) Alkali Desu lphona tion:  Re moval ofthe sul phonate g roup  by an alk ali  tr eatme nt, to gi ve u r ac il. Bisul phit e  preferen t ia lly d ea minates cyt o s i neto ur aci l  in si ngle  stranded DNA, wh e reas 5-MeC, is  ref r a cto ryt o bi su lphite-mediate d d ea m i nation.Up on PCR ampl ificatio n, uraci l is a mplifi ed as t h y mi n e whil e 5- MeC  residues  re ma i n as cy to si n es, al lowi ng  methy latedC pGst o  be distinguishe d fro m unmet h yla ted C pG s by pr e senc e of a cyt osine "C" v ersusthym ine " T" resi du e duri ngse quencing.D NA modifi catio n by bi su lphi teconver sion i s a w ell- es tab lished pr o t oc o lth a t c an b e exp lo ited  for many methodsofD NA meth yl ati o n  analy s is . Since thedet ectio n of 5-MeC b y  bis u lp h ite c onvers ion wa s first dem on strated  by F rommer et  al.1 and Cla rk 
 and_cortisol on_memory consolidation.

Institutions: Garvan Institute_of Medical_Research,_University of_NSW.

Epigenetics_describes the heritable_changes in gene_function that occur independently_to the DNA_sequence._The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to_the_DNA itself_or_the_proteins with which DNA associates._The predominant epigenetic modification of_DNA in_mammalian genomes is methylation of cytosine nucleotides (5-MeC)._DNA_methylation provides instruction_to gene expression machinery as to where and when_the gene should be expressed. The_primary target sequence_for_DNA_methylation in mammals is_5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides_are not uniformly distributed throughout the_genome, but are concentrated in regions of_repetitive genomic sequences and CpG "islands"_commonly associated with gene promoters_(Figure 1)._DNA methylation patterns are established_early in development,_modulated during_tissue specific differentiation_and disrupted in many disease states_including cancer. To_understand the biological role of DNA_methylation_and its role_in_human_disease, precise,_efficient and reproducible_methods_are required_to_detect and quantify individual 5-MeCs.
This protocol_for_bisulphite conversion is the "gold standard" for_DNA methylation analysis and_facilitates_identification and quantification of_DNA methylation at single nucleotide_resolution. The chemistry of cytosine deamination_by sodium_bisulphite involves_three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to_the 5-6 double bond of cytosine_(2) Hydrolic Deamination: hydrolytic_deamination of_the_resulting cytosine-bisulphite derivative_to_give a_uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of_the sulphonate_group by an alkali treatment, to_give uracil. Bisulphite preferentially_deaminates_cytosine to uracil in single stranded_DNA, whereas 5-MeC, is refractory to_bisulphite-mediated deamination. Upon PCR amplification,_uracil_is_amplified as thymine while 5-MeC_residues remain as cytosines, allowing methylated_CpGs to be_distinguished from unmethylated CpGs by presence of_a_cytosine "C" versus thymine "T" residue_during_sequencing.
DNA modification by bisulphite conversion is_a_well-established_protocol that can be exploited_for many methods of DNA methylation_analysis. Since the detection of 5-MeC by bisulphite conversion_was first demonstrated_by Frommer et al.1 and_Clark