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Thermo Scientific T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5'-phosphate and 3'-hydroxyl termini in duplex DNA or RNA. The enzyme repairs single-strand nicks in duplex DNA, RNA, or DNA/RNA hybrids. It also joins DNA fragments with either cohesive or blunt termini, but has no activity on single-stranded nucleic acids (see References 1, 2).
The T4 DNA Ligase requires ATP as a cofactor.
One Weiss unit** of the enzyme catalyzes the conversion of 1 nmol of [32PPi] into Norit-adsorbable form in 20 minutes at 37°C (4).
Enzyme activity is assayed in the following mixture: 66 mM Tris-HCl (pH 7.6), 6.6 mM MgCl2, 0.066 mM ATP, 10 mM DTT, 3.3 µM [32PPi].
** One Weiss unit is equivalent to approximately 200 cohesive-end ligation units. One cohesive-end ligation unit is defined as the amount of enzyme required to give 50% ligation of HindIII fragments of lambda DNA in 30 minutes at 16°C in 20 µLof the assay mixture: 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM DTT, 1 mM ATP, 25 µg/mL BSA and a 5'-DNA termini concentration of 0.12 µM (300 µg/ml).
B. H. Pheiffer et al., Polymer-stimulated ligation: enchanced blunt- or cohesive-end ligation of DNA or deoxyribo-oligonucleotides by T4 DNA ligase in polymer solutions. Nucleic Acids Res. 11, 7853-7871 (1983).
R. Rossi et al., Functional characterization of the T4 DNA Ligase: a new insight into the mechanism of action. Nucleic Acids Res. 25, 2106-2113 (1997).
A. V. Cherepanov et al., Binding of nucleotides by T4 DNA Ligase and T4 RNA Ligase: optical absorbance and fluorescence studies. Biophys. J. 81, 3545-3559 (2001).
M. Nilsson et al., RNA-templated DNA ligation for transcript analysis. Nucleic Acids Res. 29, 578-581 (2001).
B. Weiss et al., Enzymatic breakage and joining of deoxyribonucleic acid. J. Biol. Chem. 243, 4543-4555 (1968).