Specifically degrade the RNA in a DNA:RNA hybrid, without affecting DNA or unhybridized RNA, at higher reaction temperatures
- Optimal activity above 65°C and maintains activity as high as 95°C
- Highly specific for RNA in a RNA:DNA hybrid and will not digest free RNA or DNA
- Maximises digestion sensitivity and selectivity while minimising background due to nonspecific hybridization
- High-stringency hybrid selection.
- Diagnostic assays of specific target DNA sequences by isothermal probe amplification.1,2
- Transcription-based amplification methods (e.g., NASBA® method).3,4
- High-stringency mapping of mRNA structure.
- Applications that require specific hydrolysis of the RNA in a DNA:RNA hybrid.
Hybridase™ Thermostable RNase H* specifically degrades the RNA in a DNA:RNA hybrid, without affecting DNA or unhybridized RNA. In contrast to E. coli RNase H, which is rapidly inactivated at 55°C, Hybridase RNase H is active at high temperatures. It has optimal activity above 65°C and can be used at temperatures up to 95°C. The thermostability of the enzyme permits it to be used at temperatures that give the highest hybridization stringency for specific DNA:RNA heteroduplexes, maximizing sensitivity and selectivity while minimising background due to nonspecific hybridization.
Unit Definition: One unit of Hybridase RNase H results in the acid-solubilization of 1 nmol of polyadenylic acid in the presence of an equimolar concentration of polythymidylic acid in 20 minutes at 45°C under standard assay conditions.
Note: The unit assay is performed at 45°C because this is optimal for the Tm of poly(dT):poly(A). The optimal temperature for many applications may be considerably higher.
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.
Quality Control: Hybridase Thermostable RNase H is tested for RNA degradation in a RNA:DNA hybrid and for the absence of detectable exo- or endodeoxyribonuclease, and non-RNase H RNase activities.
- Duck, P. et al. (1990) BioTechniques 9, 142.
- Bekkaoui, F. et al. (1996) BioTechniques 20, 240.
- Guatelli, J.C. et al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874.
- Fahy, E. et al. (1991) PCR Methods and Applications 1, 25.