Modified RNAse H and detection of nucleic acid amplification
View Patent ↗A reversibly modified ‘hot start’ RNAse H enzyme composition is described for the improved CATACLEAVE™ 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 CATACLEAVE™ 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 CATACLEAVE™ PCR in a single reaction mix.
1. A hot start enzyme composition comprising an enzyme having an inducible RNase H activity, wherein said enzyme is reversibly modified by acylation of an amino acid residue of said enzyme or with formaldehyde.
2. The hot start enzyme composition of claim 1 , wherein said enzyme comprises an RNase H homology region having an amino acid sequence of SEQ ID NOs: 10, 11, 12, or 13.
3. The hot start enzyme composition of claim 1 , wherein said enzyme comprises an RNase H homology region having at least 70% identity with the amino acid sequence of SEQ ID NOs: 10, 11, 12, or 13.
4. The hot start enzyme composition of claim 1 wherein said enzyme comprises an RNase H homology region having at least 80% identity with the amino acid sequence of SEQ ID NOs: 10, 11, 12, or 13.
5. The hot start enzyme composition of claim 1 , said enzyme comprises an RNase H homology region having at least 90% identity with the amino acid sequence of SEQ ID NOs: 10, 11, 12, or 13.
6. The hot start enzyme composition of claim 1 , wherein said RNAse H activity is isolated from Pyrococcus furiosus, Pyrococcus horikoshi, Thermococcus litoralis, Therms thermophilus or E. coli.
7. The hot start enzyme composition of claim 1 , further comprising a polymerase activity.
8. The hot start enzyme composition of claim 7 , wherein said polymerase activity comprises a DNA polymerase activity.
9. The hot start enzyme composition of claim 8 , wherein said DNA polymerase activity is that of a thermostable DNA polymerase.
10. The hot start enzyme composition of claim 1 , further comprising a reverse transcriptase activity.
11. The hot start enzyme composition of claim 1 , wherein said RNAse H activity is heat-inducible.
12. The hot start enzyme composition of claim 11 , wherein said RNAse H activity is induced by heating a solution containing the enzyme to a temperature of about 90° C. or higher.
13. The hot start enzyme composition of claim 1 , wherein said RNAse H activity is inducible by modifying the pH of a solution containing the enzyme.
14. The hot start enzyme composition of claim 13 , wherein said RNAse H activity is induced by lowering the pH of a solution containing the enzyme to about 7.0 or lower.
15. The hot start enzyme composition of claim 14 , wherein said solution is a polymerase chain reaction sample comprising a target nucleic acid sequence.
16. The hot start enzyme composition of claim 1 , wherein said amino acid is lysine.
17. The hot start enzyme composition of claim 1 , wherein the formaldehyde is a solution containing formaldehyde at concentration of about 0.2-1% (w/v).