Assays and kits incorporating nucleic acid probes containing improved molecular switch

• US 5,312,728 A
• Filed: 05/04/1992
• Issued: 05/17/1994
• Est. Priority Date: 09/30/1988
• Status: Expired due to Term

• Current Assignee: University of Medicine and Dentistry of New Jersey (State of New Jersey)
• Original Assignee: Salk Institute for Biological Studies, The Public Health Research Institute of the City of New York, Inc.
• Inventors: Guerra, Cesar E., Buyoli, Hilda M. L., Kramer, Fred R., Tyagi, Sanjay, Lizardi, Paul M., Joyce, Gerald F., Chu, Barbara C., Orgel, Leslie E.
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US07/878,230
• Time in Patent Office: 743 Days
• Field of Search: 435/6.91, 436/501, 436/94, 536/27, 935/77, 935/78
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 1/6867 Replicase-based amplification, e.g. using Q-beta replicase
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2531/149 Replicase based amplification, e.g. Q beta replicase
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

A probe for the detection of a nucleic acid target sequence containing a molecular switch comprising three essential elements: a probe sequence of 20-60 nucleotides surrounded by switch sequences of 10-40 nucleotides which are complementary to each other, wherein the state of the switch is useful for selectively generating a detectable signal if the probe is hybridized to a target; also, assays and kits utilizing such probes.

Claims

1. A method for the detection of at least one predetermined nucleic acid target sequence in a sample containing nucleic acid and comprising the steps of:

a) adding to the sample hybridization probes comprising a molecular switch consisting essentially of three oligonucleotide sequences;

i) a probe sequence of from about 20 to about 60 nucleotides, having a 5'"'"' side and a 3'"'"' side, which probe sequence is complementary to said target sequence,

ii) a 5'"'"' sequence of from about 10 to about 40 nucleotides, including a first switch sequence of at least about 10 nucleotides, immediately adjacent to and linked to the 5'"'"' side of the probe sequence, and

iii) a 3'"'"' sequence of from about 10 to about 40 nucleotides, including a second switch sequence of at least about 10 nucleotides, immediately adjacent to and linked to the 3'"'"' side of the probe sequence, said second switch sequence being hybridized with said first switch sequence only when said probe sequence is not hybridized to said target sequence,

wherein said molecular switch includes a preselected nucleic acid sequence for selectively generating a detectable signal when the probe sequence is hybridized with said target sequence,

b) specifically hybridizing probes with said target sequence,

c) destroying the ability of probes which did not hybridize specifically with said target sequence in step b to generate a signal,

d) generating a signal from probes which did hybridize specifically with said target sequence in step b, and

e) detecting the signal in order to detect the predetermined nucleic acid target sequence.

2. A method according to claim 1 wherein said probe is a replicatable RNA.

3. A method according to claim 2 wherein step c comprises destroying with a ribonuclease the replicability of probes which did not hybridize specifically with said target sequence and wherein step d comprises exponentially replicating probes which did hybridize specifically with said target sequence.

4. A method according to claim 2 wherein probes hybridized specifically with said target sequence in step b are separated from probes which did not so hybridize prior to performing step d.

5. A method according to claim 1 comprising a quantitative determination of the amount of said target sequence in said sample, wherein the amount of probe added in step a substantially exceeds the maximum number of target sequences expected in said samples, wherein step b proceeds until virtually all target sequences hybridize with probes, wherein step c the replicability of virtually all of the probes which did not hybridize specifically with said target sequence is destroyed, wherein step d is carried out for a predetermined time, and wherein step e is quantitative.

6. A method according to claim 5 additionally comprising performing a parallel assay on a sample which does not contain said target sequence.

17. A test kit for performing an assay according to claim 1 comprising a quantity of said probes and an appropriate RNA replicase.

7. A method for the detection of at least one predetermined nucleic acid target sequence in a sample containing nucleic acid comprising the steps of

a) adding to the sample hybridization probes comprising a molecular switch consisting essentially of three oligonucleotide sequence:

i) a probe sequence of from about 20 to about 60 nucleotides, having a 5'"'"' side and a 3'"'"' side, which probe sequence is complementary to said target sequence,

ii) a 5'"'"' sequence of from about 10 to about 40 nucleotides, including a first switch sequence of at least about 10 nucleotides, immediately adjacent to and linked to the 5'"'"' side of the probe sequence, and

iii) a 3'"'"' sequence of from about 10 to about 40 nucleotides, including a second switch sequence of at least about 10 nucleotides, immediately adjacent to and linked to the 3'"'"' side of the probe sequence, said second switch sequence being hybridized with said first switch sequence only when said probe sequence is not hybridized to said target sequence,

wherein said molecular switch includes a preselected nucleic acid sequence for selectively generating a detectable signal when the probe sequence is hybridized with said target sequence, said detectable signal resulting from exponential replication of replicable RNA,

b) specifically hybridizing probes with said target sequence,

c) exponentially replicating the replicatable RNA, and

d) detecting the replication products in order to detect the predetermined nucleic acid target sequence.

8. A method according to claim 7, wherein said probe is a first DNA strand, comprising the additional step of hybridizing to the second switch sequence of said probe a second DNA strand which is a template for the transcription of a replicatable RNA prior to step c.

9. A method according to claim 7, wherein said probe is a replicatable recombinant RNA, comprising the additional step of separating probes which hybridized with target sequence in step b from probes which did not so hybridize prior to step c, and wherein the replicatable RNA of step c is said replicatable recombinant RNA.

10. A method according to claim 7 wherein said probe is an RNA strand comprising a replicatable RNA sequence extending from said second switch sequence, comprising the additional step of selectively cleaving said replicatable RNA sequence from probes which hybridized with target sequences in step b prior to step c, and wherein the replicatable RNA of step c is said replicatable RNA sequence.

11. A method according to claim 10 wherein the step of cleaving involves ribozyme cleavage.

12. A method according to claim 10 wherein the step of cleaving includes adding a ribonuclease to the sample.

13. A method according to claim 7 comprising a quantitative determination of the amount of said target sequence in said sample, wherein the amount of probe added in step a substantially exceeds the maximum number of target sequences expected in said sample, wherein step b proceeds until virtually all target hybridize with probes, wherein step c is carried out for a predetermined time, and wherein step d is quantitative.

14. A method according to claim 13 additionally comprising performing a parallel assay on a sample which does not contain said target sequence.

18. A test kit for performing an assay according to claim 7 comprising a quantity of said probes and an appropriate RNA replicase.

19. The method of claim 8 wherein the replicatable RNA is MDV-poly-RNA.

20. The method of claim 8 wherein the second DNA strand includes a promoter sequence for a DNA directed RNA polymerase.

21. The method of claim 20 wherein the exponential replication of step (c) is performed with Q-beta replicase.

22. The method of claim 9 wherein the predetermined target sequence is a gene segment.

23. The method of claim 20 wherein the promoter is for T7 RNA polymerase.

15. A method for the detection of at least one predetermined nucleic acid target sequence in a sample containing nucleic acid comprising the steps of

a) adding to the sample hybridization probes comprising a molecular switch consisting essentially of three oligonucleotides sequences:

i) a probe sequence of from about 20 to about 60 nucleotides, having a 5'"'"' side and a 3'"'"' side, which probe sequence is complementary to said target sequence,

ii) a 5'"'"' sequence of from about 10 to about 40 nucleotides, including a first switch sequence of at least about 10 nucleotides, immediately adjacent to and linked to the 5'"'"' side of the probe sequence, and

iii) a 3'"'"' sequence of from about 10 to about 40 nucleotides, including a second switch sequence of at least about 10 nucleotides, immediately adjacent to and linked to the 3'"'"' side of the probe sequence, said second switch sequence being hybridized with said first switch sequence being when said probe sequence is not hybridized to said target sequence,

wherein said molecular switch includes a preselected nucleic acid sequence for selectively generating a detectable signal from an RNA signal generator sequence of said hybridization probe when the probe sequence is hybridized with said target sequence,

b) specifically hybridizing probes with said target sequence,

c) cleaving said RNA-signal generator sequence from probes which hybridized with target sequences in step b,

d) generating an amplified signal using said RNA-signal generator, and

e) detecting said amplified signal in order to detect the predetermined nucleic acid target sequence.

16. A method according to claim 15 wherein said RNA-signal generator includes an enzyme which generates a color signal.