Nucleic acid hybridization and amplification method for detection of specific sequences in which a complementary labeled nucleic acid probe is cleaved
AssigneeUniversity of Iowa Research Foundation
InventorsJoseph A. Walder, Roxanne Y. Walder
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Abstract
A method of detection of nucleic acid (DNA or RNA) target sequence in which such a sequence serves as a cofactor for a catalytic reaction in which a complementary, labeled nucleic acid probe is cleaved such that the target sequence is released intact and can repeatedly recycle through the reaction pathway, thereby providing signal amplification.
Claims30
A method of detecting the presence of a target nucleic acid target sequence through hybridization with a substantially complementary labeled nucleic acid probe in which the probe:target nucleic acid sequence ratio is amplified through recycling of the target nucleic acid sequence, said method comprising:
- (a) hybridizing of said target nucleic acid sequence to a labeled nucleic acid probe to provide a probe:target nucleic acid sequence duplex;
- (b) cleaving only the labeled probe within the probe:target nucleic acid sequence duplex with an enzyme which causes selective probe cleavage resulting in duplex disassociation, leaving the target sequence intact;
- (c) recycling of the target nucleic acid sequence by repeating steps (a) and (b);
- and
- (d) detecting cleaved labeled probe, and thereby determining the presence of said target nucleic acid sequence.
The method of claim 14 wherein said enzyme is RNaseH.
The method of claim 83 wherein said RNAaseH enzyme is obtained from E. coli.
The method of claim 97 wherein the complementary DNA copy of the target sequence contains phosphorothioates.
Dependencies are inferred from the claim text.