US5403711A
Expired / Lapsed
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Nucleic acid hybridization and amplification method for detection of specific sequences in which a complementary labeled nucleic acid probe is cleaved
Assignee: University of Iowa Research Foundation
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Filing: 1993-07-06
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Granted: 1995-04-04
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Expires: 2013-07-06
Abstract
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.
Inventors
- 👤 Joseph A. Walder
- 👤 Roxanne Y. Walder
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-05 10:51
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/11; C12N15/113; C07; C07H; C07H21/00; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/682; C12Q1/6823; C12Q1/6827; C12Q1/683; C12Q1/6876; C12Q1/6883; C12Q1/70; C12Q1/701; C12Q1/705; C12N2310/00; C12N2310/30; C12N2310/31; C12N2310/311; C12N2310/32; C12N2310/321; C12N2310/35; C12N2310/351; C12N2310/3517; C12N2310/352; C12N2310/3521; C12N2310/3527
Claims Analysis
Total Claims: 31
Claim 1
Independent Claim
127 words
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.
Claim 2
Independent Claim
127 words
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.
Claim 3
Dependent Claim
32 words
The method of claim 1 wherein said cleaving of said labeled probe occurs at a temperature at which molecules weakly bound to sequences other than the target sequence do not remain hybridized.
Claim 4
Dependent Claim
25 words
The method of claim 2 wherein said temperature is from about 5° C. to about 25° C. below the melting temperature of the probe:target duplex.
Claim 5
Dependent Claim
13 words
The method of claim 2 wherein cleaved and unhybridized probe remains in solution.
Claim 6
Dependent Claim
19 words
The method of claim 1 wherein said nucleic acid probe is labeled with a reporter group to permit detection.
Claim 7
Dependent Claim
23 words
The method of claim 1 wherein said labeled probe is an oligonucleotide of from about 15 nucleotides to about 50 nucleotides in length.
Claim 8
Dependent Claim
26 words
The method of claim 1 wherein said enzyme will cleave only RNA hybridized in a RNA:DNA duplex and will not cleave, the target nucleic acid sequence.
Claim 9
Dependent Claim
16 words
The method of claim 1 wherein said labeled nucleic acid probe is composed entirely of RNA.
Claim 10
Dependent Claim
24 words
The method of claim 7 wherein said labeled nucleic acid probe contains one or more interspersed sequences that are not cleavable by said enzyme.
Claim 11
Dependent Claim
13 words
The method of claim 9 wherein said interspersed sequences are composed of DNA.
Claim 12
Dependent Claim
29 words
The method of claim 9 wherein said interspersed sequences are composed of nucleotide residues selected from the group consisting of phosphonates, phosphotriesters, phosphoroamidates and 2'--O alkyl and aryl ribonucleotide.
Claim 13
Dependent Claim
25 words
The method of claim 9 wherein RNA sequences within the probe cleavable by said enzyme vary from about 3 to about 15 nucleotides in length.
Claim 14
Dependent Claim
33 words
The method of claim 1 in which nonspecific cleavage of the labeled probe is suppressed by the use of single-stranded ribonuclease inhibitors selected from the group consisting of vanadate, RNAsin, and Inhibit -ACE.
Claim 15
Dependent Claim
10 words
The method of claim 14 wherein said enzyme is RNaseH.
Claim 16
Dependent Claim
14 words
The method of claim 83 wherein said RNAaseH enzyme is obtained from E. coli.
Claim 17
Dependent Claim
18 words
The method of claim 14 wherein steps (a) through (c) are carried out at a temperature above 50°.
Claim 18
Dependent Claim
28 words
The method of claim 1 wherein said detecting of cleaved probe is accomplished by separating cleavage products bearing the label from uncleaved molecules of probe remaining in solution.
Claim 19
Dependent Claim
26 words
The method of claim 17 wherein separation of cleavage products is determined by electrophoretic separation of cleavage products beating the label from uncleaved molecules of probe.
Claim 20
Dependent Claim
33 words
The method of claim 17 wherein the separation of cleavage products is determined by using strong acids to selectively precipitate uncleaved molecules of the probe, leaving cleavage products beating the label in solution.
Claim 21
Dependent Claim
115 words
The method of claim 17 wherein separation of cleavage products is accomplished with a probe modified with both an affinity label and a reporter group, and in which a solid support to which a corresponding affinity group is attached is used to adsorb molecules of intact probe, leaving cleaved fragments bearing the label in solution, said method comprising the additional steps of: adding solid support material following the hybridizing and cleaving steps; associating the affinity label attached to the probe with the corresponding affinity group on the solid support; separating the solid support from the solution containing the cleaved fragments of the probe bearing the reporter group; and detection of the reporter groups in solution.
Claim 22
Dependent Claim
30 words
The method of claim 20 in which said solid support is selected from the group consisting of polystyrene, cross-linked dextran or glass beads, cellulose, and a teflon or nylon membrane.
Claim 23
Dependent Claim
29 words
The method of claim 21 in which said solid support is magnetized and the solid support is separated from the bulk solution by the application of a magnetic field.
Claim 24
Dependent Claim
35 words
The method of claim 20 wherein said separation of cleavage products is accomplished with a nucleic acid affinity label and the corresponding affinity group attached to the solid support is a complementary nucleic acid sequence.
Claim 25
Dependent Claim
32 words
The method of claim 1 wherein said labeled probe is attached to a solid support during the steps of hybridization and cleavage, and cleaved fragments beating the label are released into solution.
Claim 26
Dependent Claim
30 words
The method of claim 24 in which said solid support is selected from the groups consisting of polystyrene, cross-linked dextran or glass beads, cellulose, and a teflon or nylon membrane.
Claim 27
Dependent Claim
26 words
The method of claim 25 in which said solid support is magnetized, and the solid support is separated from solution by application of a magnetic field.
Claim 28
Dependent Claim
90 words
The method of claim 1 for detection of a nucleic acid sequence in which the target is first used as a template for synthesis of a complementary DNA copy of the target sequence said method comprising: hybridizing of a primer to a target nucleic acid sequence 3' to the target sequence; extending of the primer with a DNA polymerase across the target sequence to produce a complementary DNA copy of the target sequence; and detecting the complementary DNA copy of the target sequence according to the method of claim 1.
Claim 29
Dependent Claim
16 words
The method of claim 97 wherein the complementary DNA copy of the target sequence contains phosphorothioates.
Claim 30
Dependent Claim
25 words
The method of claim 28 wherein said enzyme will cleave only RNA hybridized in an RNA:DNA duplex and will not cleave the complementary target sequence.
Claim 31
Dependent Claim
14 words
The method of claim 14 wherein said RNaseH is isolated from a thermophilic organism.
References Cited (Backward Citations)
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