MODIFIED RNASE H AND DETECTION OF NUCLEIC ACID AMPLIFICATION

摘要

A reversibly modified 'hot start' RNAse H enzyme composition is described for the improved CATACLEAVETM probe detection of nucleic acid sequences in a test sample. A key feature of the enzyme composition is the ability to regulate the catalytic activity of the RNAse H during the course of a reverse transcription-PCR cycle. Thus, RNAse H activity can be initially suppressed to minimize degradation of RNA:DNA primer heteroduplexes prior to reverse transcription. After cDNA synthesis is complete, RNAse H activity is induced to promote the cleavage and fluorescent detection of CATACLEAVETM probes that anneal to target DNA sequences within the reverse transcriptase-PCR products. The inducible RNAse H enzyme is amenable to high throughput applications requiring one step reverse transcriptase CATACLEAVETM PCR in a single reaction mix.