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) BioTechniques 41, 35.
  2. Shroff , H. et al. (2005) Nano Letters 5(7), 1509.
  3. Lin, C. et al. (2006) Angewandte Chemie 118, 7699.
  4. Korlach, J. et al. (2008) Proc. Natl. Acad. Sci. USA 105, 1176.
  5. McArthur, M. and Bibb, M.J. (2008) Proc. Natl. Acad. Sci. USA 105, 1020.
  6. Shroff , H. et al. (2008) Biophysical Journal 94, 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.

*Covered by intellectual property rights licensed to Epicentre.


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