3rd July 2024

CircLigase- 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™ ssDNA Ligase* is a thermostable ATP-dependent ligase 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
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 >30 bases is circularized by CircLigase enzyme. Under standard
reaction conditions, virtually no linear concatamers or circular concatamers
are produced. In addition to its activity on ssDNA, CircLigase enzyme also
has activity in ligating a single-stranded nucleic acid having a 3´-hydroxyl
ribonucleotide and a 5´-phosphorylated ribonucleotide or deoxyribonucleotide.

Unit Definition:One unit of CircLigase enzyme converts 1 pmol of a linear 5´-phosphorylated CircLigase 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:0.5 M MOPS (pH 7.5), 0.1 M KCl, 50 mM MgCl2, and 10 mM DTT. The Reaction Buffer does not containATP or MnCl2which must be added to the reaction at final concentrations of 0.05 mM ATP and 2.5 mM MnCl2. A 2.5-mM solution of ATP and a 50-mM solution of MnCl2are included.

Quality Control:CircLigase 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™ 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™ 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
CL4111K 100 U/µl 1,000 Units
CL4115K 100 U/µl 5,000 Units
Contents: CircLigase™ ssDNA Ligase, CircLigase™ 10X Reaction Buffer, 1 mM ATP, 50 mM MnCl2, CircLigase™ ssDNA Control Oligo, Sterile Water.
*Covered by intellectual property rights licensed to Epicentre.