3rd July 2024

CircLigase- II ssDNA Ligase

Applications
  • Production of ssDNA templates for rolling-circle replication or rolling-circle transcription experiments.
  • Production of ssDNA templates for RNA polymerase and RNA polymerase inhibitor assays.

CircLigase™ II ssDNA Ligase* is a thermostable enzyme that catalyzes
intramolecular ligation (i.e. circularization) of ssDNA templates having a
5´-phosphate and a 3´-hydroxyl group. In contrast to T4 DNA Ligase
and Ampligase®DNA Ligase, which ligate DNA ends that are annealed
adjacent to each other on a complementary DNA sequence, CircLigase II ssDNA
Ligase ligates ends of ssDNA in the absence of a complementary sequence. The
enzyme is therefore useful for making circular ssDNA molecules from linear
ssDNA. Circular ssDNA molecules can be used as substrates for rolling-circle
replication or rolling-circle transcription.

Linear ssDNA of >15 bases, including cDNA, is circularized by CircLigase
II enzyme. Under standard reaction conditions, virtually no linear concatamers
or circular concatamers are produced. In addition to its activity on ssDNA,
CircLigase II enzyme also has activity in ligating a single-stranded nucleic
acid having a 3´-hydroxyl ribonucleotide and a 5´-phosphorylated
ribonucleotide or deoxyribonucleotide.

CircLigase II has greater activity and a new Reaction Buffer for improved
ligation efficiency.

Unit Definition:One unit of CircLigase II enzyme converts 1 pmol of a linear 5´-phosphorylated CircLigase II Standard 55-mer Oligo into an exonuclease I-resistant circular form in 1 hour at 60°C under standard assay conditions.Storage Buffer:50% glycerol containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, and 0.1% Triton®X-100.10X Reaction Buffer:0.33 M Tris-Acetate (pH 7.5), 0.66 M potassium acetate and 5 mm DTT.

Quality Control:CircLigase II ssDNA Ligase is free of detectable phosphatase, DNA exo- and endonuclease, and RNase activities.

Product Citations

  1. Polidoros, A.N.et al.(2006)BioTechniques41, 35.
  2. Shroff , H.et al.(2005)Nano Letters5(7), 1509.
  3. Lin, C.et al.(2006)Angewandte Chemie118, 7699.
  4. Korlach, J.et al.(2008)Proc. Natl. Acad. Sci. USA105, 1176.
  5. McArthur, M. and Bibb, M.J. (2008)Proc. Natl. Acad. Sci.USA105, 1020.
  6. Shroff , H.et al.(2008)Biophysical Journal94, 2179.
  7. Kuhn, H. and Frank-Kamenetskii, M.D. (2008)Nucleic Acids Res.36, e40.
  8. Murakami, T.et. al.(2008)Nucleic Acids Res.Doi:10.1093/nar/gkn1014

Figure 1. CircLigase™ II ssDNA Ligase converts linear ssDNA to circular ssDNA. A 71-base ssDNA oligo was converted to a circular DNA form in a reaction containing CircLigase™ II ssDNA Ligase and ATP. Lane 1, DNA markers. Lane 2, 71-base ssDNA. Lane 3, circularization proceeds through an adenylated intermediate. Lane 4, the closed circular nature of the reaction product was confirmed by treating the reaction with exonuclease I, which specifically digests linear DNA.

Ordering

Catalog No. Concentration Size
CL9021K           100 U/µl 1,000 Units
CL9025K           100 U/µl 5,000 Units
Contents: CircLigase™ II ssDNA Ligase, CircLigase™ II 10X Reaction Buffer, 50 mM MnCl2, CircLigase™ ssDNA Control Oligo, Betaine, Sterile Water.

*Covered by intellectual property rights licensed to Epicentre.