Synthesise "RNA" transcripts that also contain standard or modified dNTPs using this mutant T7 RNA polymerase
- Generate "RNA" transcripts with both rNMP/2´-dNMP or rNMP/2´-modified-NMP composition
- Create modified "RNA" transcripts that are resistant to RNase A-type RNases
- Use for a variety of applications where "RNA" with incorporated dNTPs is beneficial
T7 R&DNA™ Polymerase* is a recombinant mutant form of T7 RNA polymerase (Y639F mutant), in which Tyr-639 has been replaced by Phe.1,2 This mutation enables T7 R&DNA Polymerase to incorporate 2´-deoxyribonucleoside triphosphates (dNTPs) into full-length transcripts much more efficiently than the corresponding wild-type enzyme,1while retaining the same catalytic activity for incorporation of canonical NTPs, and the same high promoter specificity as wild-type T7 RNA Polymerase. The ability of this mutant enzyme to incorporate certain 2´-modified-dNTPs (including but not limited to dNTPs having a 2´-fluorine or 2´-amino group) enables efficient in vitro synthesis of transcripts composed of mixed rNMP/2´-dNMP or rNMP/2´- modified-dNMP from any DNA template that is downstream of the T7 RNA polymerase promoter. Such modified transcripts are useful for many applications.
Note: Complete substitution of one 2´-dNTP (or one 2´-modified dNTP) for a canonical rNTP in a T7 R&DNA Polymerase reaction results in a slight decrease in yield. Additional substitutions of 2´-dNTPs (or 2´-modified-dNTPs) for canonical rNTPs will further reduce yield of the transcript produced. Substitution with all four 2´-dNTPs (or 2´-modified dNTPs) will result in extremely low yields of transcript and is NOT RECOMMENDED.
Unit Definition: One unit of T7 R&DNA Polymerase catalyzes the incorporation of 1 nmol of ribonucleoside triphosphate into RNA in 1 hour at 37°C under standard assay conditions using a DNA template with the T7 promoter.
Storage Buffer: 50% glycerol containing 50 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 1.0 mM DTT, 0.1 mM EDTA, and 0.1% Triton® X-100.
5X Transcription Buffer: 0.2 M Tris-HCl (pH 7.5), 50 mM NaCl, 30 mM MgCl2, and 10 mM spermidine.
- Sousa, R. and Padilla, R. (1995) EMBO J. 14, 4609.
- U.S. Patent Nos. 5,849,546 and 6,107,037.