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Epicentre Forum 1 (4)
T4 RNA Ligase catalyzes ATP-dependent joining of nucleic
acids that have a 5´-phosphorylated terminus (donor) and a 3´-hydroxylated
terminus (acceptor).1 Although the
enzyme is active on RNA, DNA, and numerous nucleotide derivatives,2-5 RNA
is the preferred acceptor molecule for the enzyme. When an oligodeoxynucleotide
is the acceptor, ligation efficiency is greatly increased by adding ribonucleotides
to its 3´-hydroxyl terminus, particularly if adenine is the base
in these added ribonucleotides.2
Applications for T4 RNA Ligase include:
-
mapping and sequencing of RNA 5´- and 3´-termini2,6-9
-
5´-end labeling of RNA with oligonucleotides for
cDNA synthesis6-8
-
construction of mixed DNA-RNA molecules 2,5,10,11
The enzyme is certified free of contaminating RNase and
DNA exo- and endonuclease activities.
Figure 1 demonstrates the use of Epicentre's T4 RNA Ligase
in intermolecular ligation of a mixed DNA-RNA acceptor oligo with a DNA
donor oligo.
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Figure 1. Intermolecular ligation of oligonucleotides using
T4 RNA Ligase. A 30-mer oligodeoxynucleotide (donor) was 5´-phosphorylated
using T4 Polynucleotide Kinase (Epicentre) and then purified by
polyacrylamide gel electrophoresis (PAGE). A 24-mer mixed DNA-RNA
oligo with 23 deoxyribonucleotides and one adenine ribonucleoside
at the 3´-hydroxyl terminus was the acceptor molecule. The
reaction mixture contained 2 µg of the 24-mer acceptor, 1 µg
of the 30-mer donor, 5 µM ATP, and 10 U of Epicentre's T4
RNA Ligase in 1X T4 RNA Ligase Buffer (supplied with the enzyme).
Following incubation of the reaction mixture at 37°C for 2hr,
10 µl was electrophoresed on a 20% TBE PAGE gel, and the
gel was stained with ethidium bromide. Lane 1, 24-mer DNA-RNA acceptor.
Lane 2, 30-mer DNA donor. Lane 3, 24-mer acceptor and 30-mer donor;
no enzyme. Lane 4, complete reaction. The ligated product in Lane
4 is indicated by the arrow. |
- Silber, et al. (1972) Proc. Natl. Acad. Sci. USA 69: 3009.
- Uhlenbeck and Gumport (1982) in The Enzymes Vol 15, Academic Press,
New York, 31.
- Ohtsuka et al. (1978) Biochemistry 17: 4894.
- England et al. (1977) Proc. Natl. Acad. Sci. USA 74: 4839.
- Walker et al. (1975) Proc. Natl. Acad. Sci. USA 72: 122.
- Volloch et al. (1994) Nuc. Acids Res. 22: 2507.
- Fromont-Racine et al. (1993) Nuc. Acids Res. 21: 1683.
- Mandel et al. (1991) BioTechniques 10: 485.
- England and Uhlenbeck (1978) Nature 275: 560.
- Romaniuk and Uhlenbeck (1983) Methods Enzymol. 100: 52.
- Tessier et al. (1986) Anal. Biochem. 158: 171.
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