Q-Beta Replicase (Qβ Replicase) is an RNA-directed RNA polymerase that is responsible for replication of the coliphage Q-Beta RNA genome. Various aspects of the enzyme are presented in a review by Blumenthal and Carmichael.1 It is composed of four subunits, one of which is encoded by the Q-Beta phage and three by the E. coli host. EPICENTRE's Q-Beta Replicase is purified from E. coli containing a plasmid that expresses the phage-encoded subunit. All four enzyme subunits are present in equal proportions (Fig. 1).
In vitro, Q-Beta Replicase can utilize other RNA molecules besides the Q-Beta phage RNA as templates. These include various subgenomic variant RNA molecules that were found in Q-Beta Replicase reactions, such as midivariant (MDV) RNAs, and other smaller variant RNAs. In addition, it can also use other "non-natural" templates such as poly(rC), RNA primed with an oligoribonucleotide, or RNA tailed with several C residues. Recombinant RNA molecules consisting of RNA sequences embedded within midivariant MDV-1 RNA can also be replicated by Q-Beta Replicase.2 The enzyme uses both strands of MDV-1 RNA or recombinant MDV-1 RNAs as templates, and synthesis of product is exponential so long as the enzyme is in excess. Large amplification of recombinant sequences can be achieved in a short time under isothermal conditions.
Unit Definition: One unit of Q-Beta Replicase catalyzes the incorporation of 1 nmole of rGTP into poly(rG) in 10 minutes at 30°C using 20 ng/µl of poly(rC) as a template in a reaction mixture containing 33 mM Tris-acetate (pH 7.5), 66 mM potassium acetate, 10 mM magnesium acetate, 0.5 mM DTT, and 1 mM rGTP.
Storage Buffer & Conditions: 50% glycerol containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA, and 0.1% Triton X-100. Store at -20°C in a freezer without a defrost cycle.
10X TA Reaction Buffer (available separately): 330 mM Tris-acetate (pH 7.5), 660 mM potassium acetate, 100 mM magnesium acetate, and 5 mM DTT.
Figure 1. SDS-PAGE of Qβ Replicase (lane 3) along with E. coli RNA polymerase (lane 1) and molecular weight markers (lane 2). The subunit names of E. coli RNA polymerase (β´, β, σ and α) and Qβ Replicase (S1, II, Tu, and Ts) are indicated.
Quality Control: The physical purity of Q-Beta Replicase is greater than 95%, as determined by SDS polyacrylamide gel electrophoresis. All four subunits are present in equal amounts. In order to minimize the possibility of contamination of the Q-Beta Replicase by Q-Beta templates, EPICENTRE uses only poly(rC) as a template for determining activity. Enzymatic assays of Q-Beta Replicase activity without added template produce no detectable RNA by gel analysis, thus ruling out contamination with RNA that is exponentially amplifiable by the enzyme. Gel analysis also indicates the absence of contamination by E. coli DNA-dependent RNA polymerase. The enzyme is free of detectable contaminating exo- and endonuclease and RNase activities.
- Blumenthal, T and Carmichael, GG. (1979) Ann. Rev. Biochem. 48, 525.
- Lizardi, PM et al. (1988) Biotechnology 6, 1197.
|Enzyme only. 10X TA Reaction Buffer and NTPs are available separately.|