Methods for detection of a target nucleic acid using multi-subunit probes

• US 20030148310A1
• Filed: 07/17/2002
• Published: 08/07/2003
• Est. Priority Date: 07/17/2001
• Status: Active Grant

• Current Assignee: Stratagene California (Agilent Technologies, Inc.)
• Original Assignee: Stratagene California (Agilent Technologies, Inc.)
• Inventors: Sorge, Joseph
• Granted Patent: US 7,183,052 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2537/137 a displacement step
• CPC Class Codes: C12Q 2537/1373 Displacement by a nucleic acid
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide

Abstract

The invention relates to a method of generating a signal indicative of the presence of a target nucleic acid sequence in a sample, comprising forming a complex by incubating a sample comprising a target nucleic acid sequence with a probe comprising a first and second subunit, and/or an upstream primer, and binding the probe to the target nucleic acid such that the first and second subunits dissociate to release the first subunit and generate a signal. In certain embodiments, the upstream primer is extended with a nucleic acid polymerase to displace at least a portion of the first subunit of the probe from the target nucleic acid strand and dissociate the first and second subunits to release the first subunit of the probe and generate a signal.

Claims

1. A method of generating a signal indicative of the presence of a target nucleic acid sequence in a sample, comprising

forming a detection complex by incubating a sample comprising a target nucleic acid sequence and a probe, wherein said probe comprises a first and a second subunit, and

binding said probe to said target nucleic acid sequence, wherein said first subunit of said probe dissociates from said second subunit of said probe to generate a signal,

wherein said binding is performed at a binding temperature and said first subunit of said probe does not dissociate from said second subunit of said probe when not bound to said target nucleic acid sequence at or below said binding temperature, and

wherein generation of said signal is indicative of the presence of a target nucleic acid sequence in said sample.

3. The method of claim 1 or 2, wherein said signal is detected or measured, and wherein said detecting and/or measuring the signal comprises detecting and/or measuring the amount of said released first subunit.

6. The method of claim 1, 2, 3, or 4 wherein said first subunit of said probe further comprises at least one labeled moiety capable of providing a signal.

7. The method of claim 1, 2, 3, or 4 wherein a detection complex is formed comprising a pair of interactive signal generating labeled moieties effectively positioned on said probe to quench the generation of a detectable signal when the probe is not bound to said target nucleic acid or when said first subunit of said probe is not dissociated from said second subunit of said probe.

8. The method of claim 7 wherein said pair of interactive signal generating moieties comprises a quencher moiety and a fluorescent moiety.

2. A method of generating a signal indicative of the presence of a target nucleic acid sequence in a sample, comprising

a) forming a detection complex by incubating a sample comprising a target nucleic acid sequence, an upstream primer, and a downstream probe, wherein said downstream probe comprises a first and a second subunit, and binding said probe to said target nucleic acid sequence,

b) subjecting said detection complex to a nucleic acid polymerization activity under conditions which permit extension of said upstream primer by polymerization of a nucleic acid strand, and displacement of said first subunit of said probe from said target nucleic acid sequence by said nucleic acid strand, such that said first subunit dissociates from said second subunit of said probe and is released to generate a signal,

wherein said binding is performed at a binding temperature and said first subunit of said probe does not dissociate from said second subunit of said probe when not bound to said target nucleic acid sequence at or below said binding temperature, and

wherein said polymerization is performed at a polymerization temperature and said first subunit of said probe does not dissociate from said second subunit of said probe when not displaced by said nucleic acid strand at or below said polymerization temperature, and

wherein generation of said signal is indicative of the presence of a target nucleic acid sequence in said sample.

4. A method of detecting or measuring a target nucleic acid sequence comprising the steps of:

a) forming a detection complex by incubating a sample comprising a target nucleic acid sequence with a probe, wherein said probe comprises a first and a second subunit, and binding said probe to said target nucleic acid sequence to dissociate said first subunit of said probe from said second subunit of said probe, to generate a signal,

wherein said binding is performed at a binding temperature and said first subunit of said probe does not dissociate from said second subunit of said probe when not bound to said target nucleic acid sequence at or below said binding temperature; and

b) detecting and/or measuring the amount of said released first subunit as an indication of the presence of the target sequence in the sample.

5. A method of detecting or measuring a target nucleic acid sequence comprising the steps of:

a) forming a detection complex by incubating a sample comprising a target nucleic acid sequence, an upstream primer, and a downstream probe, wherein said probe comprises a first and a second subunit, and binding said probe to said target nucleic acid sequence,

b) subjecting said detection complex to a nucleic acid polymerization activity under conditions which permit extension of said upstream primer by polymerization of a nucleic acid strand, and displacement of said first subunit of said probe from said target nucleic acid sequence by said nucleic acid strand, such that said first subunit dissociates from said second subunit of said probe and is released to generate a signal,

wherein said binding is performed at a binding temperature and said first subunit of said probe does not dissociate from said second subunit of said probe when not bound to said target nucleic acid sequence at or below said binding temperature,

wherein said polymerization is performed at a polymerization temperature and said first subunit of said probe does not dissociate from said second subunit of said probe when not displaced by said nucleic acid strand, at or below said polymerization temperature, and

detecting and/or measuring the amount of said released first subunit as an indication of the presence of the target sequence in the sample.

9. A polymerase chain reaction process for detecting a target nucleic acid sequence in a sample comprising:

(a) providing a detection complex comprising a probe, wherein said probe comprises a first and a second subunit, a set of oligonucleotide primers wherein a first primer contains a sequence complementary to a region in one strand of said 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

(b) 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 said target nucleic acid sequence, (ii) extending the primers wherein said nucleic acid polymerase synthesizes a primer extension product, and thereby dissociates said first subunit of said probe from said second subunit of said probe thereby creating detectable, released labeled first subunits of said probe;

wherein said amplification is performed at an amplification temperature and said first subunit of said probe does not dissociate from said second subunit of said probe when not bound to said target nucleic acid sequence at or below said amplification temperature; and

(c) detecting and/or measuring the amount of released, labeled first subunit of said probe as an indicator of the presence of the target sequence in the sample.

11. The polymerase chain reaction process of claim 9 wherein said oligonucleotide primers of step b are oriented such that the forward primer is located upstream of said detection complex and the reverse primer is located downstream of said detection complex.

10. A polymerase chain reaction process for detecting a target nucleic acid sequence in a sample comprising:

(a) providing a detection complex comprising a probe, wherein said probe comprises a first and a second subunit, a set of oligonucleotide primers wherein a first primer contains a sequence complementary to a region in one strand of said 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

(b) 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 said target nucleic acid sequence, (ii) extending the primers wherein said nucleic acid polymerase synthesizes a primer extension product, and (iii) displacement of said first subunit of said probe from said target nucleic acid sequence by said primer extension product, such that said first subunit dissociates from said second subunit of said probe thereby creating detectable, released labeled first subunits of said probe;

wherein said amplification is performed at an amplification temperature and said first subunit of said probe does not dissociate from said second subunit of said probe when not bound to said target nucleic acid sequence, or when not displaced from said target nucleic acid sequence by said primer extension product, at or below said amplification temperature; and

(c) detecting and/or measuring the amount of released, labeled first subunit of said probe as an indication of the presence of the target sequence in the sample.

12. The polymerase chain reaction process of claim 9 or 10 wherein the nucleic acid polymerase is thermostable.

13. The polymerase chain reaction process of claim 9 or 10 wherein said labeled detection complex is formed by the addition of a labeled first subunit of said probe capable of providing a signal and a second subunit of said probe.

14. The polymerase chain reaction process of claim 9 or 10 wherein said detection complex is formed comprising a pair of interactive signal generating labeled moieties effectively positioned on said probe to quench the generation of a detectable signal when said probe is not bound to said target nucleic acid or when said first subunit of said probe is not dissociated from said second subunit of said probe.

15. The polymerase chain reaction process of claim 14 wherein said pair of interactive signal generating moieties comprises a quencher moiety and a fluorescent moiety.

16. A method of forming a detection complex comprising the steps of:

(a) providing a target nucleic acid sequence,

(b) providing a probe, wherein said probe comprises a first and a second subunit, and

(c) binding said target nucleic acid sequence and said probe and;

wherein said binding is performed at a binding temperature and said first subunit of said probe does not dissociate from said second subunit of said probe when not bound to said target nucleic acid sequence at or below said binding temperature.

17. A method of forming a detection complex comprising the steps of:

(a) providing a target nucleic acid sequence,

(b) providing an upstream primer complementary to said target nucleic acid sequence,

(c) providing a probe, wherein said probe comprises a first and a second subunit, and

(c) binding said target nucleic acid sequence, said upstream primer and said probe and;

wherein said binding is performed at a binding temperature and said first subunit of said probe does not dissociate from said second subunit of said probe when not bound to said target nucleic acid sequence at or below said binding temperature.

18. A composition comprising a target nucleic acid sequence, and a probe, wherein said probe comprises a first and a second subunit, and

wherein said probe and said target nucleic acid can bind to form a detection complex and wherein said binding is performed at a binding temperature, and wherein said first subunit of said probe does not dissociate from said second subunit of said probe when not bound to said target nucleic acid sequence at or below said binding temperature.

19. A composition comprising a target nucleic acid sequence, a probe, wherein said probe comprises a first and a second subunit, an upstream primer and a nucleic acid polymerization activity,

wherein said probe, said primer and said target nucleic acid can bind to form a detection complex and wherein said binding is performed at a binding temperature, and wherein said first subunit of said probe does not dissociate from said second subunit of said probe when not bound to said target nucleic acid sequence at or below said binding temperature.

20. A kit for generating a signal indicative of the presence of a target nucleic acid sequence in a sample, comprising a probe, wherein said probe comprises a first and a second subunit, and

packaging means thereof,

wherein said probe can bind to a target nucleic acid sequence to form a detection complex;

and

wherein said binding is performed at a binding temperature, and wherein said first subunit of said probe does not dissociate from said second subunit of said probe when not bound to said target nucleic acid sequence at or below said binding temperature.

22. The kit of claim 20 or 21, wherein said probe comprises a labeled first subunit of said probe and a second subunit of said probe.

23. The kit of claim 20 or 21, wherein said first subunit of said probe comprises a pair of interactive signal generating labeled moieties effectively positioned to quench the generation of a detectable signal when said probe is not bound to said target nucleic acid or when said first subunit of said probe is not dissociated from said second subunit of said probe.

24. The kit of claim 23, wherein said pair of interactive signal generating moieties comprises a quencher moiety and a fluorescent moiety.

21. A kit for generating a signal indicative of the presence of a target nucleic acid sequence in a sample comprising an upstream primer, and a downstream probe, wherein said probe comprises a first and a second subunit, and a nucleic acid polymerization activity, and

packaging means thereof,

wherein said primer and said,probe can bind to a target nucleic acid sequence to form a detection complex;

and

wherein said binding is performed at a binding temperature, and wherein said first subunit of said probe does not dissociate from at least said second subunit of said probe when not bound to said target nucleic acid sequence at or below said binding temperature.