What is claimed is:
1. A method for detecting a nuclease-mediated cleavage of a target nucleic acid, which method comprises:
(a) hybridizing a target nucleic acid to a fluorescently labeled oligonucleotide probe complementary to the target nucleic acid and containing a flourophor at one terminus and a quenching group at the other terminus, wherein (i) when the probe is unhybridized to the target nucleic acid, the probe adopts a conformation that places the flourophor and quencher in such proximity that the quencher quenches the flourescent signal of the flourophor and (ii) formation of the probe-target hybrid causes sufficient separation of the flourophor and quencher to reduce quenching of the flourescent signal of the flourophor;
(b) contacting the probe-target hybrid with an agent having nuclease activity in an amount sufficient to selectively cleave the target nucleic acid and thereby release the intact probe; and
(c) detecting the release of the probe by measuring a decrease in the flourescent signal of the flourophor as compared to the signal of the probe-target hybrid.
2. The method of claim 1, wherein the agent is an enzyme having an RNase H activity.
3. The method of claim 2, wherein the agent is selected from the group consisting of HIV reverse transcriptase, E. coli RNase H1, E. coli RNase H2, Human RNase H1, Human RNase H2, hammerhead ribozyme, HBV reverse transcriptase, and integrase.
4. The method of claim 1, wherein the probe is DNA, and the target is the DNA:RNA hybrid substrate.
5. The method of claim 1, wherein the probe is at least 18 nucleotides in length.
6. The method of claim 1, wherein the probe, when unhybridized to the target nucleic acid, adopts a hairpin secondary structure conformation that brings the fluorophor and quencher into proximity.
7. The method of claim 1, wherein the nuclease reaction is performed in the presence of a compound, wherein a difference in the rate of the decrease in the flourescent signal of the flourophor during the nuclease reaction, as compared to the decrease observed when the same reaction is conducted in the absence of the compound, is indicative of the ability of the compound to either inhibit or enhance the nuclease activity of the agent.
8. The method of claim 1, which further comprises monitoring the flourescent signal of the flourophor during the nuclease reaction.
9. A method for measuring a RNase H activity of an agent, which method comprises:
(a) hybridizing a target RNA to a fluorescently labeled oligodesoxyribonucleotide probe complementary to the target RNA and containing a flourophor at one terminus and a quenching at the other terminus, wherein (i) when the probe is unhybridized to the target RNA, the probe adopts a conformation that places the flourophor and quencher in such proximity that the quencher quenches the flourescent signal of the flourophor and (ii) formation of the probe-target hybrid causes sufficient separation of the flourophor and quencher to reduce quenching of the flourescent signal of the flourophor;
(b) contacting the probe-target hybrid with the agent in an amount sufficient to selectively cleave the target RNA and thereby release the intact probe; and
(c) measuring a decrease in the flourescent signal of the flourophor as compared to the signal of the probe-target hybrid.
10. The method of claim 9, wherein the agent is an enzyme having an RNase H activity.
11. The method of claim 10, wherein the agent is selected from the group consisting of HIV reverse transcriptase, E. coli RNase H1, E. coli RNase H2, Human RNase H1, Human RNase H2, hammerhead ribozyme, HBV reverse transcriptase, and integrase.
12. The method of claim 9, wherein the probe is at least 18 nucleotides in length.
13. The method of claim 9, wherein the probe, when unhybridized to the target RNA, adopts a hairpin secondary structure conformation that brings the fluorophor and quencher into proximity.
14. The method of claim 9, wherein the RNase H-mediated reaction is performed in the presence of a compound, wherein a difference in the rate of the decrease in the flourescent signal of the flourophor during the RNase H-mediated reaction, as compared to the decrease observed when the same reaction is conducted in the absence of the compound, is indicative of the ability of the compound to either inhibit or enhance the RNase H activity of the agent.
15. The method of claim 9, which further comprises monitoring the flourescent signal of the flourophor during the RNase H-mediated reaction.
16. A method of screening for a modulator of the nuclease activity of an agent, which method comprises:
(a) hybridizing a target nucleic acid to a fluorescently labeled oligonucleotide probe complementary to the target nucleic acid and containing a flourophor at one terminus and a quenching group at the other terminus, wherein (i) when the probe is unhybridized to the target nucleic acid, the probe adopts a conformation that places the flourophor and quencher in such proximity that the quencher quenches the flourescent signal of the flourophor and (ii) formation of the probe-target hybrid causes sufficient separation of the flourophor and quencher to reduce quenching of the flourescent signal of the flourophor;
(b) preparing two samples containing the probe-target hybrid;
(c) contacting the probe-target hybrid of a first sample with the agent in an amount sufficient to selectively cleave the target nucleic acid and thereby release the intact probe;
(d) contacting the probe-target hybrid of a second sample with the agent in an amount sufficient to selectively cleave the target nucleic acid and thereby release the intact probe in the presence of a candidate compound, which is being tested for its ability to modulate the nuclease activity of the agent;
(e) detecting the release of the probe in each sample by measuring a decrease in the flourescent signal of the flourophor as compared to the signal of the probe-target hybrid; and
(f) comparing the rate of the decrease in the flourescent signal of the flourophor in the two samples, wherein a difference in the rate of the decrease in the flourescent signal of the flourophor during the nuclease reaction in the two samples is indicative of the ability of the compound to either inhibit or enhance the nuclease activity of the agent.
17. The method of claim 16, wherein a greater extent or relative rate of decrease of the flourescent signal of the flourophor in the second sample compared to the first sample indicates that the candidate compound is an agent agonist.
18. The method of claim 16, wherein a lesser extent or relative rate of decrease of the flourescent signal of the flourophor in the second sample compared to the first sample indicates that the candidate compound is an agent antagonist.
19. A kit for measuring a nuclease activity of an agent, comprising a target nucleic acid and a fluorescently labeled oligonucleotide probe complementary to the target nucleic acid and containing a flourophor at one terminus and a quencher at the other terminus, wherein (i) when the probe is unhybridized to the target nucleic acid, the probe adopts a conformation that places the flourophor and quencher in such proximity that the quencher quenches the flourescent signal of the flourophor and (ii) formation of the probe-target hybrid causes sufficient separation of the flourophor and quencher to reduce quenching of the flourescent signal of the flourophor.
20. The kit of claim 19, wherein the probe is at least 18 nucleotides in length.
21. The kit of claim 19, wherein the probe, when unhybridized to the target nucleic acid, adopts a hairpin secondary structure conformation that brings the fluorophor and quencher into proximity.
22. The kit of claim 19, wherein the probe is DNA, and the target nucleic acid is DNA:RNA hybrid substrate.
23. The kit of claim 19, further comprising the agent.
24. The kit of claim 23, wherein the agent is is selected from the group consisting of RNase H, reverse transcriptase, E. coli RNase H1 and H2, Human RNase H1 and H2, hammerhead ribozymes, HBV reverse transcriptase, and integrase.
25. The kit of claim 23, wherein the reverse transcriptase is HIV reverse transcriptase.
26. An assay mixture for measuring a nuclease activity of an agent, comprising a target nucleic acid and a fluorescently labeled oligonucleotide probe complementary to the target nucleic acid and containing a flourophor at one terminus and a quenching group at the other terminus, wherein (i) when the probe is unhybridized to the target nucleic acid, the probe adopts a conformation that places the flourophor and quencher in such proximity that the quencher quenches the flourescent signal of the flourophor and (ii) formation of the probe-target hybrid causes sufficient separation of the flourophor and quencher to reduce quenching of the flourescent signal of the flourophor.
27. The assay mixture of claim 26, wherein the probe is DNA, and the target nucleic acid is RNA.
28. The assay mixture of claim 26, wherein the probe and the target nucleic acid are hybridized to each other to form a probe-target hybrid.
29. The assay mixture of claim 28, further comprising the agent.
30. The assay mixture of claim 29, wherein the agent is selected from the group consisting of RNase H, reverse transcriptase, E. coli RNase H1 and H2, Human RNase H1 and H2, hammerhead ribozymes, HBV reverse transcriptase, and integrase.
31. The assay mixture of claim 30, wherein the reverse transcriptase is HIV reverse transcriptase.
The claims below are in addition to those above.
All refrences to claim(s) which appear below refer to the numbering after this setence.
1. An output control method for a consumable electrode arc welding power source in which an output of the welding power source is controlled based on an amplified error value between a predetermined voltage setting value and a welding voltage,
wherein an entire application range of a welding current is segmentalized into a plurality of current zones, a norm welding voltage value is set for each of the current zones, a welding current and a welding voltage are detected at each of minute cycles, one of the current zones which corresponds to a value of the detected welding current is selected, a fluctuation range is calculated with the norm welding voltage value of the selected current zone as a center value, a welding voltage limit value is calculated while the detected value of the welding voltage is limited to within the fluctuation range, and the amplified error value is calculated using the voltage setting value and the welding voltage limit value.
2. The method according to claim 1, wherein a moving average of the welding voltage limit values is obtained for each of the current zones for the calculation of the norm welding voltage value for each of the current zones.
3. An output control method for a consumable electrode arc welding power source in which an output of a welding power source is controlled based on an amplified error value between a predetermined voltage setting value and a welding voltage,
wherein a norm characteristic of an arc characteristic curve which indicates a relation between a welding current and a welding voltage during arc generation is set, a welding current and a welding voltage are detected at each of minute cycles, a norm welding voltage value which corresponds to a detected value of the welding current is calculated on the norm characteristic, a fluctuation range is calculated with the norm welding voltage value as a center value, a welding voltage limit value is calculated while the detected value of the welding voltage is limited to within the fluctuation range, and the amplified error value is calculated using the voltage setting value and the welding voltage limit value.
4. The method according to claim 3, wherein an approximating curve is calculated for each of predetermined calculation cycles while welding is performed using operational points data constituted from the detected value of the welding current and the corresponding welding voltage limit value in each of the minute cycles, and the approximating curve is set as the norm characteristic.
5. The method according to claim 1, wherein an output voltage which is a voltage at a point on a output path in the welding power source before the reactor mounted on the output path is detected, a deviation between the detected value of the welding voltage and the welding voltage limit value is calculated, a voltage control setting value is calculated by adding the deviation to the voltage setting value, and the amplified error value is calculated using the voltage control setting value and the detected value of the output voltage.
6. The method according to claim 1, wherein each of the norm welding voltage values in all of the current zones is modified based on a change amount of the voltage setting value in the case that the voltage setting value changes.
7. The method according to claim 3, wherein the norm characteristic is modified toward a direction of a welding voltage axis based on a change amount of the voltage setting value in the case that the voltage setting value changes.
8. The method according to claim 3, wherein the norm characteristic is set differently for a plurality of potions to be welded.