Methods for detection of a target nucleic acid sequence
DCFirst Claim
1. A method of generating a signal indicative of the presence of a target nucleic acid sequence in a sample, comprising forming a cleavage structure comprising duplex and single-stranded nucleic acid by incubating a sample comprising a target nucleic acid sequence with a nucleic acid polymerase and cleaving said cleavage structure with a FEN nuclease to generate a signal, wherein generation of said signal is indicative of the presence of a target nucleic acid sequence in said sample.
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Abstract
The invention relates to a method of generating a signal indicative of the presence of a target nucleic acid sequence in a sample, where the method includes forming a cleavage structure by incubating a sample containing a target nucleic acid sequence with a nucleic acid polymerase and cleaving the cleavage structure with a FEN nuclease to generate a cleaved nucleic acid fragment. The invention also relates to methods of detecting or measuring a target nucleic acid sequence, where the method includes forming a cleavage structure by incubating a target nucleic acid sequence with a nucleic acid polymerase, cleaving the cleavage structure with a FEN nuclease and detecting or measuring the release of a fragment.
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Citations
31 Claims
- 1. A method of generating a signal indicative of the presence of a target nucleic acid sequence in a sample, comprising forming a cleavage structure comprising duplex and single-stranded nucleic acid by incubating a sample comprising a target nucleic acid sequence with a nucleic acid polymerase and cleaving said cleavage structure with a FEN nuclease to generate a signal, wherein generation of said signal is indicative of the presence of a target nucleic acid sequence in said sample.
- 3. A method of detecting or measuring a target nucleic acid sequence comprising forming a cleavage structure comprising duplex and single-stranded nucleic acid by incubating a sample comprising a target nucleic acid sequence with a nucleic acid polymerase, cleaving said cleavage structure with a FEN nuclease to release a nucleic acid fragment and detecting and/or measuring the release of said fragment as an indication of the presence of the target sequence in the sample.
- 13. A polymerase chain reaction process for detecting a target nucleic acid sequence in a sample comprising providing a cleavage structure comprising duplex and single-stranded nucleic acid, providing a set of oligonucleotide primers wherein a first primer contains a sequence complementary to a region in one strand of the target nucleic acid sequence and primes the synthesis of a complementary DNA strand, and a second primer contains a sequence complementary to a region in a second strand of the target nucleic acid sequence and primes the synthesis of a complementary DNA strand and amplifying the target nucleic acid sequence employing a nucleic acid polymerase as a template-dependent polymerizing agent under conditions which are permissive for PCR cycling steps of (i) annealing of primers required for amplification to a template nucleic acid sequence contained within the target nucleic acid sequence, (ii) extending the primers wherein said nucleic acid polymerase synthesizes a primer extension product, and (iii) cleaving said cleavage structure employing a FEN nuclease as a cleavage agent for release of labeled fragments from said cleavage structure thereby creating detectable labeled fragments and detecting and/or measuring the release of labeled fragments as an indication of the presence of the target sequence in the sample.
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23. A polymerase chain reaction process for simultaneously forming a cleavage structure comprising duplex and single-stranded nucleic acid, amplifying a target nucleic acid sequence in a sample and cleaving said cleavage structure comprising:
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(a) providing an upstream oligonucleotide primer complementary to a region in one strand of the target nucleic acid sequence and a downstream labeled probe complementary to a region in the same strand of the target nucleic acid sequence, wherein the upstream primer contains a sequence complementary to a region in one strand of the target nucleic acid sequence and primes the synthesis of a complementary DNA strand, and the downstream probe contains a sequence complementary to a region in a second strand of the target nucleic acid sequence and primes the synthesis of a complementary DNA strand; and
(b) detecting a nucleic acid which is produced in a reaction comprising amplification of said target nucleic acid sequence and cleavage thereof wherein a nucleic acid polymerase is a template-dependent polymerizing agent under conditions which are permissive for PCR cycling steps of (i) annealing of primers to a target nucleic acid sequence, (ii) extending the primers of step (a) wherein said nucleic acid polymerase synthesizes primer extension products, and wherein the primer extension product of the primer of step (a) partially displaces the downstream probe of step (a) to form a cleavage structure comprising duplex and single-stranded nucleic acid; and
(iii) cleaving said cleavage structure employing a FEN nuclease as a cleavage agent for release of labeled fragments from said cleavage structure thereby creating detectable labeled fragments.- View Dependent Claims (24)
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25. A method of forming a cleavage structure comprising providing a target nucleic acid sequence, providing an upstream primer complementary to said target nucleic acid sequence, providing a downstream probe complementary to said target nucleic acid sequence, extending the 3′
- end of the upstream primer with a nucleic acid polymerase substantially lacking 5′
to 3′
exonuclease activity; and
displacing the 5′
end of the downstream probe.
- end of the upstream primer with a nucleic acid polymerase substantially lacking 5′
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26. A kit for generating a signal indicative of the presence of a target nucleic acid sequence in a sample, comprising a FEN nuclease, a suitable buffer and a nucleic acid polymerase that substantially lacks 5′
- to 3′
exonuclease activity, wherein said FEN nuclease, said buffer and said nucleic acid polymerase are in the same composition. - View Dependent Claims (27, 28, 29)
- to 3′
- 30. A composition comprising a nucleic acid polymerase, a FEN nuclease, and a nucleic acid primer.
Specification