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

• US 7,183,052 B2
• Filed: 07/17/2002
• Issued: 02/27/2007
• Est. Priority Date: 07/17/2001
• Status: Expired due to Fees

• Current Assignee: Stratagene California (Agilent Technologies, Inc.)
• Original Assignee: Stratagene California (Agilent Technologies, Inc.)
• Inventors: Sorge, Joseph
• Primary Examiner(s): Horlick; Kenneth R.
• Application Number:
  US10/196,842
• Publication Number:
  US 20030148310A1
• Time in Patent Office: 1,686 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/810, 536/24.3
• US Class Current: 435/6.12
• 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 further comprises;

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 the probe is associated with said second subunit of the probe, and

wherein said first or second subunit of said probe comprises said pair of interactive signal generating moieties, 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 and wherein said first or second subunit of said probe comprising said pair of interactive signal generating moieties undergoes a conformational change 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.

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 comprising a first, second and third region, an upstream primer, and a downstream probe, wherein said downstream probe comprises a first and a second subunit, wherein said first and second subunit each comprise a first and second region, wherein said second region of said first subunit is complementary to said first region of said second subunit and said second region of said first subunit binds to said first region of said second subunit when not bound to said target nucleic acid sequence;

and

wherein said first region of said first subunit is complementary to said first region of said target nucleic acid sequence, said second region of said first subunit is complementary to said second region of said target nucleic acid sequence, said first region of said second subunit is complementary to said second region of said target nucleic acid sequence and said second region of said second subunit is complementary to said third region of said target nucleic acid sequence;

and binding said probe to said target nucleic acid sequence, such that said first region of said first subunit binds to said first region of said target nucleic acid sequence, said first region of said second subunit binds to said second region of said target nucleic acid sequence and said second region of said second subunit binds to said third region of said target nucleic acid sequence, and said first and second subunits dissociate from each other;

b) extending said upstream primer by polymerization of a nucleic acid strand by a nucleic acid polymerization activity, and displacing said first subunit of said probe from said target nucleic acid sequence by said nucleic acid strand, such that said first subunit of said probe dissociates from said target nucleic acid sequence 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.

6. The method of claim 2 or 5 wherein said first subunit of said probe further comprises at least one labeled moiety capable of providing a signal.

7. The method of claim 2 or 5 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.

25. The method of claim 2, wherein only one subunit of said probe is displaced from said target nucleic acid sequence by said nucleic acid strand in step (b).

28. The method of claim 2, wherein the 3′

terminus of the subunit of the probe that is bound to said target nucleic acid sequence is blocked.

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 further comprises;

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 the probe is associated with said second subunit of the probe, and

wherein said first or second subunit of said probe comprises said pair of interactive signal generating moieties, 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, and wherein said first or second subunit of said probe comprising said pair of interactive signal generating moieties undergoes a conformational change 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 a first, second and third region 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 wherein said first and second subunit each comprise a first and second region, wherein said second region of said first subunit is complementary to said first region of said second subunit and said second region of said first subunit binds to said first region of said second subunit when not bound to said target nucleic acid sequence; and

wherein said first region of said first subunit is complementary to said first region of said target nucleic acid sequence, said second region of said first subunit is complementary to said second region of said target nucleic acid sequence, said first region of said second subunit is complementary to said second region of said target nucleic acid sequence and said second region of said second subunit is complementary to said third region of said target nucleic acid sequence; and

binding said probe to said target nucleic acid sequence such that said first region of said first subunit binds to said first region of said target nucleic acid sequence, said first region of said second subunit binds to said second region of said target nucleic acid sequence and said second region of said second subunit binds to said third region of said target nucleic acid sequence, and said first and second subunits dissociate from each other;

b) extending said upstream primer by polymerization of a nucleic acid strand by a nucleic acid polymerization activity, and displacing said first subunit of said probe from said target nucleic acid sequence by said nucleic acid strand, such that said first subunit of said probe dissociates from said target nucleic acid sequence 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.

26. The method of claim 5, wherein only one subunit of said probe is displaced from said target nucleic acid sequence by said nucleic acid strand in step (b).

29. The method of claim 5, wherein the 3′

terminus of the subunit of the probe that is bound to said target nucleic acid sequence is blocked.

9. A polymerase chain reaction process for detecting a target nucleic acid sequence comprising a first, second and third region in a sample comprising:

(a) providing a detection complex comprising a probe, wherein said probe comprises a first and a second subunit wherein said first and second subunit each comprise a first and second region, wherein said second region of said first subunit is complementary to said first region of said second subunit and said second region of said first subunit binds to said first region of said second subunit when not bound to said target nucleic acid sequence; and

wherein said first region of said first subunit is complementary to said first region of said target nucleic acid sequence, said second region of said first subunit is complementary to said second region of said target nucleic acid sequence, said first region of said second subunit is complementary to said second region of said target nucleic acid sequence and said second region of said second subunit is complementary to said third region of said target nucleic acid sequence;

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;

wherein said probe binds to said target nucleic acid sequence such that said first region of said first subunit binds to said first region of said target nucleic acid sequence, said first region of said second subunit binds to said second region of said target nucleic acid sequence and said second region of said second subunit binds to said third region of said target nucleic acid sequence, and said first and second subunits dissociate from each other; and

(b) amplifying the target nucleic acid sequence by the steps of (i) annealing of primers required for amplification to a template nucleic acid sequence contained within said target nucleic acid sequence, and (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 target nucleic acid sequence 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.

30. The polymerase chain reaction process of claim 9, wherein a subunit of the probe binds said target nucleic acid and wherein the 3′

terminus of the subunit of the probe that is bound to said target nucleic acid sequence is blocked.

10. A polymerase chain reaction process for detecting a target nucleic acid sequence comprising a first, second and third region in a sample comprising:

(a) providing a detection complex comprising a probe, wherein said probe comprises a first and a second subunit wherein said first and second subunit each comprise a first and second region, wherein said second region of said first subunit is complementary to said first region of said second subunit and said second region of said first subunit binds to said first region of said second subunit when not bound to said target nucleic acid sequence; and

wherein said first region of said first subunit is complementary to said first region of said target nucleic acid sequence, said second region of said first subunit is complementary to said second region of said target nucleic acid sequence, said first region of said second subunit is complementary to said second region of said target nucleic acid sequence and said second region of said second subunit is complementary to said third region of said target nucleic acid sequence;

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 wherein said probe binds to said target nucleic acid sequence such that said first region of said first subunit binds to said first region of said target nucleic acid sequence, said first region of said second subunit binds to said second region of said target nucleic acid sequence and said second region of said second subunit binds to said third region of said target nucleic acid sequence, and said first and second subunits dissociate from each other; and

(b) amplifying the target nucleic acid sequence by the 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) displacing 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 target nucleic acid sequence 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.

27. The polymerase chain reaction process of claim 10, wherein only one subunit of said probe is displaced from said target nucleic acid sequence by said primer extension product in step (b).

31. The polymerase chain reaction process of claim 10, wherein a subunit of the probe binds said target nucleic acid and wherein the 3′

terminus of the subunit of the probe that is bound to said target nucleic acid sequence is blocked.

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 further comprises;

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 the probe is associated with said second subunit of the probe, and

wherein said first or second subunit of said probe comprises said pair of interactive signal generating moieties, 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 comprising a first, second and third region,

(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, wherein each of said first and second subunits comprises a 5′

terminus and a 3′

terminus, wherein said first and second subunit each comprise a first and second region, wherein said second region of said first subunit is complementary to said first region of said second subunit and said second region of said first subunit binds to said first region of said second subunit when not bound to said target nucleic acid sequence; and

wherein said first region of said first subunit is complementary to said first region of said target nucleic acid sequence, said second region of said first subunit is complementary to said second region of said target nucleic acid sequence, said first region of said second subunit is complementary to said second region of said target nucleic acid sequence and said second region of said second subunit is complementary to said third region of said target nucleic acid sequence; and

(d) binding said target nucleic acid sequence, said upstream primer and said probe such that said first region of said first subunit binds to said first region of said target nucleic acid sequence, said first region of said second subunit binds to said second region of said target nucleic acid sequence and said second region of said second subunit binds to said third region of said target nucleic acid sequence, and said first and second subunits dissociate from each other; 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, and wherein said 3′

terminus of said first and/or second subunit of said probe that is bound to said target nucleic acid sequence is blocked.

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

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 the probe is associated with said second subunit of the probe,

wherein said first or second subunit of said probe comprises said pair of interactive signal generating moieties,

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 comprising a first, second and third region, a probe, wherein said probe comprises a first and a second subunit, wherein each of said first and second subunits comprises a 5′

terminus and a 3′

terminus, wherein said first and second subunit each comprise a first and second region, wherein said second region of said first subunit is complementary to said first region of said second subunit and said second region of said first subunit binds to said first region of said second subunit when not bound to said target nucleic acid sequence; and

wherein said first region of said first subunit is complementary to said first region of said target nucleic acid sequence, said second region of said first subunit is complementary to said second region of said target nucleic acid sequence, said first region of said second subunit is complementary to said second region of said target nucleic acid sequence and said second region of said second subunit is complementary to said third region of said target nucleic acid sequence, 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, wherein said probe binds to said target nucleic acid sequence such that said first region of said first subunit binds to said first region of said target nucleic acid sequence, said first region of said second subunit binds to said second region of said target nucleic acid sequence and said second region of said second subunit binds to said third region of said target nucleic acid sequence, and said first and second subunits dissociate from each other;

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, and wherein said 3′

terminus of said first and/or second subunit of said probe that is bound to said target nucleic acid sequence is blocked.

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 further comprises:

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 the probe is associated with said second subunit of the probe,

wherein said first or second subunit of said probe comprises said pair of interactive signal generating moieties, 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.

21. A kit for generating a signal indicative of the presence of a target nucleic acid sequence comprising a first, second and third region in a sample comprising an upstream primer, and a downstream probe, wherein said probe comprises a first and a second subunit, wherein each of said first and second subunits comprises a 5′

terminus and a 3′

terminus, wherein said first and second subunit each comprise a first and second region, wherein said second region of said first subunit is complementary to said first region of said second subunit and said second region of said first subunit binds to said first region of said second subunit when not bound to said target nucleic acid sequence; and

wherein said first region of said first subunit is complementary to said first region of said target nucleic acid sequence, said second region of said first subunit is complementary to said second region of said target nucleic acid sequence, said first region of said second subunit is complementary to said second region of said target nucleic acid sequence and said second region of said second subunit is complementary to said third region of said target nucleic acid sequence, 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, wherein said probe binds to said target nucleic acid sequence such that said first region of said first subunit binds to said first region of said target nucleic acid sequence, said first region of said second subunit binds to said second region of said target nucleic acid sequence and said second region of said second subunit binds to said third region of said target nucleic acid sequence, and said first and second subunits dissociate from each other; 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, and wherein said 3′

terminus of said first and/or second subunit of said probe that is bound to said target nucleic acid sequence is blocked.

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

23. The kit of claim 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.