Tth 111 Reverse Transcriptase

Thermus thermopilus Reverse Transcriptase catalyzes the reverse transcription of RNA to cDNA at elevated temperatures in the presence of Mn²⁺.

Source: Thermus thermophilus

Description:

· Suitable for high temperature synthesis of DNA (1).

· Synthesizes cDNA from RNA template (2).

· Results in greater specificity of primer hybridization and extension of RNA.

· Can reverse transcribe at elevated temperatures (2).

· Minimizes problems with strong secondary structure of RNA.

· Used for efficient PCR of DNA, containing problematic secondary structures.

· Applicable to RT-PCR; the same enzyme is used for both reverse transcription and following amplification of obtained cDNA template (2).

· Resistant to amplification inhibitors present in template DNA isolated from problematic samples (3,4).

Unit Definition:one unit is the amount of enzyme required to incorporate 1 nmol of dTTP into acid-insoluble form in 10 min at 50°C.

Storage Conditions: Store at –20°C.

Storage Buffer: 50 mM Tris-HCl (pH 7.5 at 22°C), 5 mM dithiothreitol, 0.1 mM EDTA, 50% (v/v) glycerol and stabilizers.

Assay Conditions: 40 mM Tris-HCl (pH 8.5 at 22°C), 1 mM MnCl₂, 1 mg/ml bovine serum albumin, 10 mM dithiothreitol, 0.5 mM [α-³²P] dTTP and 0.4 mM poly(A)·(dT)₅₀. Incubation is at 50°C for 10 min in a reaction volume of 50 µl.

Quality Control: All preparations are assayed for contaminating endonuclease, exonuclease, nonspecific RNase and single- and double-stranded DNase activities.

References:

1. Wang, A. M., Doyle, M.V. and Mark, D.F. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9717-9721.

2. Myers, T. W., Gelfanld, D. H. (1991) Biochemistry 30, 7661-7666.

3. Kather, H. L., Schwartz, I. (1994) Biotechniques 16, 84-92.

4. Poddar, S. K., Sawyer, M. H., Connor, J. D. (1998) J. Med. Microbiol. 47, 1131-1135.