ChlorellavirusPBCV-1DNAligase(ChlorellavirusDNAligase,alsoknownasSplintR™LigasesoldbyNEB),isanATP-dependentDNAligase.PBCV-1DNAligaseefficientlycatalyzestheligationofadjacent,single-strandedDNAsplintedbyacomplementaryRNAstrand.ThisunusualactivitycanbeusedtocharacterizemiRNAs,RNAsandSNPs,e.g.sensitive(sub-nanomolarrange)detectionofspecificRNAbyligation.Inaddition,PBCV-1DNAligasemaybeusedforenrichmentofRNAfornext-generationsequencing.

PBCV-1DNAligaseenzymeisactiveoverabroadrangeofATPconcentrations(10µM–1mM)andpH(6.5–9).OptimalactivityisobservedusingMg2+>5mM,andapHbetween7.5and8.0.Theactivityisenhancedatelevatedtemperatures(upto37°C),andbysupplementationwith5mMMn2+.Thereactionisinhibitedbysaltconcentrationswhenconcentrationis>100mM.

Theenzymetoleratesallbasepaircombinationsattheligationjunction,butispartiallyinhibitedbydC/GanddG/Cbasepairsatthedonor(phosphorylated)sideligationjunction,particularlywhenthe+2basewasalsoaC/Gbasepair.

• LigationofssDNAsplintedbycomplementaryRNAsequences
• DetectionofRNAusingligationofDNAprobes
• SNPorsplicevariantdetection
• RASL-seq

• ChlorellavirusPBCV-1DNAligase,10,000unitsinstoragebuffer:10mMTris-HCl,300mMNaCl,1mMDTT,0.1mMEDTA,50%Glycerol,pH7.4@25°C
• 10XLigationBuffer:250mMTris-HCI,50mMMgCl₂,5mMDTT,10mMATP,pH7.6@25°C

Theabsenceofendo-,exodeoxyribonucleases,andribonucleasesconfirmedbyappropriatequalitytests.