T7 R&DNA™ Polymerase

Applications

• Synthesis of “RNA” transcripts of mixed rNMP/2´-dNMP or rNMP/2´-modified-NMP composition.
• Synthesis of modified “RNA” transcripts that are resistant to RNase A-type RNases.

T7 R&DNA™ Polymerase* is a recombinant mutant form of T7 RNA polymerase (Y639F mutant), in which Tyr-639 has been replaced by Phe.1,2 This mutation enables T7 R&DNA Polymerase to incorporate 2´-deoxyribonucleoside triphosphates (dNTPs) into full-length transcripts much more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,1 while 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.

References

1. Sousa, R. and Padilla, R. (1995) EMBO J. 14, 4609.
2. U.S. Patent Nos. 5,849,546 and 6,107,037.

Ordering