Description:

Single-StrandBindingProtein(SSB)bindstosingle-strandedDNAwithhighaffinityandalsobindstoRNAanddouble-strandedDNAwithloweraffinity(1).Invivo,itstABIlizestransientlyformedssDNAandplaysanimportantroleinDNAreplication,recombinationandrepair(2).Invitro,SSBproteinshavebeenusedtodestabilizesecondarystructuresinDNAandtoincreasetheprocessivityofDNApolymerasesinseveralmolecularBIOLOGyapplications:SSBsimprovetheyieldandefficiencyofreversetranscriptionreactionsduringRT-PCRaswellasincreasetheyieldofPCRproducts(3-9).

ETSSB(ExtremeThermostableSSB)isasingle-strandedDNAbindingproteinisolatedfromahyperthermophilicmicroorganism,anditisaflagshipenhancerofthePrimerNavigatorproductseries.Itremainsfullyactiveafterincubationat95ºCfor60min.Duetotheextremethermostability,ETSSBcanbeusedinapplicationsthatrequireextremelyhightemperatureconditions,suchasnucleicacidamplificationandsequencing.

Source:

Applications:

ImprovetheyieldofmultiplexPCR(Fig.1)andmultiplexHDA(Fig.2)
ImprovetheprocessivityofDNApolymerase(10)
StabilizationandmarkingofssDNAstructure(11)
IncreasetheyieldandspecificityofPCRreactions(5-9)
IncreasetheyieldandprocessivityofRTduringRT-PCR(3,4)
ImproveDNAsequencingthroughregionswithstrongsecondarystructure(8)
EnhancetheRecAactivityforssDNAbindingandstrandtrasfer(12,13)

StorageConditions:

StorageBuffer:
20mMTris-HCl
200mMNaCl
1mMEDTA
0.5mMDTT
50%Glycerol
pH7.5@RT

StorageTemperature:
-20ºC

QualityControl:

QualityAssuranceStatement:
ETSSBispurifiedfreeofcontaminatingendonucleasesandexonucleases.Eachlotistestedforsingle-strand,DNA-dependentATPaseactivityandisvisuallydeterminedtobe>95%pureonanSDS-polyacrylamidegel.

ExonucleaseActivity:
Incubationof20µgETSSBfor4hoursat37°Cin50µlreactionbuffercontaining50mMpotassiumacetate,20mMTris-acetate,10mMmagnesiumacetateand1mMdithiothreitol,pH7.9@25°C,with1µgofamixtureofsingleanddouble-stranded[³H]E.coliDNA(200,000cpm/µg)released<0.05%ofthetotalrADIoactivity.

EndonucleaseAssay:
Incubationof10µgETSSBfor4hoursat37°Cin50µlreactionbuffercontaining50mMpotassiumacetate,20mMTris-acetate,10mMmagnesiumacetateand1mMdithiothreitol,pH7.9@25°C,with1µgΦX174RFIDNAgave<5%conversiontoRFII.

NucleaseActivity:
Incubationof20µgETSSBfor16hoursat37°Cin50µlofreactionbuffercontaining50mMpotassiumacetate,20mMTris-acetate,10mMmagnesiumacetateand1mMdithiothreitol,pH7.9@25°C,with1µgλDNAyieldedaclearandsharpbandonanagarosegel.

References:

1. Coleman,J.E.&Oakley,J.L.(1980)CRCCritRevBiochem7,247-89.
2. Chase,J.W.&Williams,K.R.(1986)AnnuRevBiochem55,103-36.
3. Baugh,L.R.,Hill,A.A.,Brown,E.L.&Hunter,C.P.(2001)NucleicAcidsRes29,E29.
4. Villalva,C.,Touriol,C.,Seurat,P.,Trempat,P.,Delsol,G.&Brousset,P.(2001)Biotechniques31,81-3,86.
5. Schwarz,K.,Hansen-Hagge,T.&Bartram,C.(1990)NucleicAcidsRes18,1079.
6. Chou,Q.(1992)NucleicAcidsRes20,4371.
7. Oshima,R.G.(1992)Biotechniques13,188.
8. Rapley,R.(1994)MolBiotechnol2,295-8.
9. Olszewski,M.,Rebala,K.,Szczerkowska,Z.&Kur,J.(2005)MolCellProbes19,203-5.
10. Myers,T.W.&Romano,L.J.(1988)JBiolChem263,17006-15.
11. Delius,H.,Mantell,N.J.&Alberts,B.(1972)JMolBiol67,341-50.
12. Reddy,M.S.,Vaze,M.B.,Madhusudan,K.&Muniyappa,K.(2000)Biochemistry39,14250-62.
13. West,S.C.,Cassuto,E.&Howard-Flanders,P.(1982)MolGenGenet186,333-8.