Description:
ThePyrophosphatase,inorganic(PPase)catalyzesthehydrolysisofinorganicpyrophosphatetotwoorthophosphates.Theenzymerequiresadivalentmetalcation,withMg2+conferringthehighestactivity.
Application:
-HighyieldsynthesisofRNAbyinvitrotranscription(1,2)
-DNApolymerizationreactions:preventingaccumulationofpyrophosphate(3,4)
-RemovalofcontaminantPPiinreagentsusedforSNPgenotypingbymethodsbasedonthedetectionofpyrophosphate(5)
Source:
E.colicellswithaclonedppageneofSacharomycescerevisiae.
UnitDefinition:
Oneunitistheamountofenzymethatwillgenerate1µmolofphosphateperminutefrominorganicpyrophosphateunderstandardreactionconditions(a10minutereactionat25°Cin100mMTris-HCL,[pH7.2],2mMMgCl2and2mMPPiinareactionvolumeof0.5ml).
StorageConditions:
20mMTris-HCl
100mMKCl
1mMDithiothreitol
0.1mMEDTA
50%Glycerol
pH8.0@25°C
RecommendedStorageCondition:-20°C
References:
1.Cooperman,B.S.,Themechanismofactionofyeastinorganicpyrophosphatase,Meth.Enzymol.,87,526-548,1982.
2.Cunningham,P.R.andOfengand,J.,UseofinorganicpyrophostasetoimprovetheyieldofinvitrotranscriptionreactionscatalyzedbyT7RNApolymerase,Biotechniques,9,713-714,1990.
3.Tabor,S.,Richardson,C.C.,DNAsequenceanalysiswithamodifiedbacteriophageT7DNApolymerase.Effectofpyrophosphorolysisandmetalions,J.Biol.Chem.,265,8322-8328,1990.
4.Dean,B.F.,etal.,RapidamplificationofplasmidandphageDNAusingphi29DNApolymeraseandmultiply-primedRollingCircleamplification,GenomeRes.,11,1095-1099,2001.
5.Zhou,G.H.,etal.,Quantitativedetectionofsinglenucleotidepolymorphismsforapooledsamplebyabioluminometricassaycoupledwithmodifiedprimerextensionreactions(BAMPER),NucleicAcidsRes.,29,E93,2001. |
