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This enzyme (once known as polynucleotide ligase [12,13]) joins nicks in double-stranded duplexes, including (RNA:RNA)/DNA hybrids [14,15]. T4 DNA ligase presents several advantages over the other methods for ligating RNA [11]. First, its low Km for polynucleotide duplexes (from 10–8 to 10–7 M) means that the ligation reactions can proceed in reasonable times even when substrate concentrations are in the submicromolar to micromolar range. Second, because this enzyme is specific for nicks in doublestranded duplexes, production of undesired side products is kept to a minimum.

Luckily, T4 DNA ligase discriminates against such longer 5' substrates and does not incorporate them into ligated products (5). 2. For efficient ligation to occur, it is crucial that the concentration of the bridging cDNA template be equal to or intermediate between those of the RNA ligation substrates. If excess template is present, then individual substrate molecules can hybridize to different template molecules and not have a ligation partner. For this reason, any attempt to “drive” the hybridization by adding excess template is futile.

1984) Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 12, 7035–7056. 2. Krieg, P. A. and Melton, D. A. (1987) In vitro synthesis with SP6 RNA polymerase. Methods Enzymol. 155, 397–415. 3. Gurevich, V. V. (1996) Use of bacteriophage RNA polymerase in RNA synthesis. Methods Enzymol. 275, 382–397. 4. Milligan, J. , Groebe, D. , Witherell, G. , and Uhlenbeck, O. C. (1987) Oligoribo-nucleotide synthesis using T7 RNA polymerase and synthetic DNA template.

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