Fluorometric assay for detecting nucleic acid cleavage
AssigneeGeorgetown University
InventorsMyun Ki Han, S. Paul Lee, Jack Chirikjian
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Abstract
A method of detecting an enzyme-mediated DNA cleavage reaction in a fluorometric assay is provided. The method can be used to detect DNA cleavage caused by restriction endonucleases, retroviral integrase enzymes, DNases, RNases, or enzymes utilized in other strand separating processes in molecular biology.
Claims35
A method for continuously detecting a specific nucleic acid sequence that is produced during a process that results in amplification of a specific DNA or RNA sequence, said process comprising:
- (a) obtaining a fluorescent labeled oligonucleotide containing a nucleic acid sequence recognized by an enzyme that facilitates nucleic acid cleavage or ligation;
- (b) contacting said oligonucleotide with said enzyme that facilitates nucleic acid ligation or cleavage during a process that results in amplification of said specific DNA or RNA sequence;
- and
- (c) continuously detecting said ligation or cleavage reaction by detecting a change in fluorescence intensity during said amplification reaction.
A method for continuously detecting a specific nucleic acid sequence by a fluorometric assay comprising the following steps:
- (a) obtaining an oligonucleotide that is labeled with a fluorescent acceptor and donor pair, wherein (1) at least one of said acceptor and donor are attached to said oligonucleotide by a linker that inhibits quenching upon binding of said oligonucleotide to another nucleic acid sequence, and (2) said oligonucleotide is specifically recognized by an enzyme that effects nucleic acid ligation or cleavage of a specific nucleic sequence;
- (b) contacting said oligonucleotide with said enzyme that effects nucleic acid ligation or cleavage of said oligonucleotide;
- and
- (c) continuously detecting said ligation or cleavage reaction by monitoring fluorescence intensity during said ligation or cleavage reaction.
A method for continuously detecting a specific nucleic acid sequence that is produced during a process that results in amplification of a specific DNA or RNA sequence, said process comprising:
- (a) obtaining an oligonucleotide that is labeled with a fluorescent acceptor and donor pair, wherein (1) at least one of said acceptor and donor are attached to said oligonucleotide by a linker that inhibits quenching or fluorescence upon biding of said oligonucleotide to another nucleic acid sequence, and (2) said oligonucleotide is specifically recognized by an enzyme that effects nucleic acid ligation or cleavage of a specific nucleic acid sequence;
- (b) contacting said oligonucleotide with said enzyme that effects nucleic acid ligation or cleavage during a process that results in amplification of said specific nucleic acid sequence;
- and
- (c) continuously detecting said ligation or cleavage reaction, and thereby detecting said specific nucleic acid sequence produced during said amplification reaction, by monitoring fluorescence intensity as said amplification reaction proceeds.
Dependencies are inferred from the claim text.