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
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.
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.