NEB/LpnPI/R0663S/200 units

Description:

LpnPI,anEpiMark^®validatedproduct,isamodification-dependentendonucleasewhichrecognizesC^mCDGsitesandgeneratesadouble-strandedDNAbreakonthe3´sideofthemodifiedcytosineatN₁₀/N₁₄.RecognizedcytosinemodificationsincludeC5-methylation(5-mC)andC5-hydroxymethylation(5-hmC)(1). 

ThisenzymeisprovidedwithanEnzymeActivatorSolutionwhichmaybeusedforefficientdigestionbyLpnPI.

ThemostcommonepigeneticmodificationsfoundineukaryoticorganismsaremethylationmarksatCpGorCHGsites.AsubsetofthesemodifiedsitesarerecognizedandcleavedbyLpnPI. 

AtfullymethylatedCpGsites: 
5´...C^mC GG ...3´
3´...G G ^mC C...5´

orCHGsites: 
5´... C^mCD GG ...3´
3´...GGH^mCC...5´

H=AorCorT(notG)
D=AorGorT(notC) 

LpnPIrecognizeseachhemi-methylatedsiteindividuallyandcleavesbidirectionallytogenerate32-baseor31-basefragments,respectively.Thesefragmentscontainthecentralmethylatedsiteandhave4-base5´overhangsateachend.LpnPIdoesnotcleaveunmodifiedDNA.

ProductSource

An E.coli strainthatcarriesthesyntheticLpnPIgenefrom Legionellapneumophila speciesPhiladelphia1.

ReagentsSupplied

Thefollowingreagentsaresuppliedwiththisproduct:

	Storeat(°C)	Concentration
CutSmart®Buffer	-20	10X
EnzymeActivatorSolution		30X

Notes:

ThisenzymehasbeenusedforepigeneticanalysisinwhichtargetDNAcontainingCpGsitesmethylatedinbothstrandsiscleavedbyLpnPItogenerate32bpfragmentscontainingthemethylatedCpGsites.Theisolated32bpfragmentscanbesequencedtoreveal5-mCmodificationsites(Cohen-Karnietal.,(2011)PNAS108:11040-11045).Useofexcessenzymeinhibitscleavage.OptimizationoftheamountofenzymeneededforcompletedigestionmayberequiredforeachsubstrateDNA.Staractivity,includingdigestionofnon-methylatedDNAsubstrates,mayresultfromnon-optimalreactionconditionssuchasextendeddigestiontime,highenzymeoractivatorconcentrationoraglycerolconcentrationof>5%.Optimizationoftheamountofenzymeneededtoachieveefficientspecificcutting,whileavoidingstaractivity,mayberequiredforeachsubstrateDNA.Optimalspecificityofcleavageat5-mCsitescanbeachievedbykeepingthemolarratioofLpnPIto5-mCtargetsitesbetween0.1:1to1:1inthepresenceof1XEnzymeActivatorSolutionanddigestingforupto60minutes.(ThemolarityofLpnPIis~2.9μM).