Method for detecting a nucleic acid involving the production of a triggering RNA and transcription amplification

• US 5,871,914 A
• Filed: 04/09/1996
• Issued: 02/16/1999
• Est. Priority Date: 06/03/1993
• Status: Expired due to Fees

• Current Assignee: Intelligene Ltd.
• Original Assignee: Intelligene Ltd.
• Inventors: Nathan, Asher
• Primary Examiner(s): ARTHUR, LISA BENNETT
• Application Number:
  US08/556,940
• Time in Patent Office: 1,043 Days
• Field of Search: 435/91.21, 435/6, 435/91.2, 935/77, 935/78, 536/24.33, 536/24.3
• US Class Current: 435/6.14
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm

Abstract

A method for detecting the presence of an assayed nucleic acid in a sample is an essentially two-stage procedure. In a first stage the sample is reacted in a manner which gives rise to the production of a triggering RNA where the sample contains the assays sequence. In the second stage, the reaction product is incubated under appropriate conditions with an amplification reagent ensemble whereby, in the presence of triggering RNA a large amount of a nucleic product is obtained. The detection of this product thus indicates the presence of the assayed sequence in the sample.

Claims

1. A method for detecting the presence of an assayed nucleic acid sequence in a sample, comprising the steps of:

(a) reacting the sample with a detection ensemble comprising;

a first DNA molecule having a promoter sequence at the 3'"'"' end and a 5'"'"' end sequence which is complementary to the 5'"'"' end portion of the assayed nucleic acid sequence;

a second DNA molecule comprising a single-stranded 3'"'"' end sequence being complementary to a 3'"'"' end portion of the assayed nucleic acid sequence, and further comprising a sequence, at the 5'"'"' end which can be transcribed into a triggering RNA sequence capable of initiating a reaction in an appropriate transcription system in which an RNA molecule having a signal RNA sequence is being produced;

wherein the 3'"'"' end sequence of the second DNA molecule and the 5'"'"' end sequence of the first DNA are complementary to at least part of said assayed nucleic acid sequence leaving an intermediary portion in the assayed nucleic acid having no complementary counterpart in either the first or the second DNA molecules, in which case the detection ensemble further comprises

a third DNA sequence being complementary to said intermediate portion;

(b) incubating under conditions to allow hybridization of said first DNA molecule and said second DNA molecule and were present also said third DNA molecule with said assayed nucleic acid sequence, and optionally adding a ligase to allow ligation of adjacent ends of said first, second and third DNA molecules;

(c) adding transcription reagents comprising a DNA dependent-RNA polymerase and RNA nucleotides and incubating under conditions to allow the formation of RNA transcripts having said triggering RNA sequence;

(d) contacting the RNA transcripts with an RNA amplification ensemble, a priori, lacking the triggering RNA, in which a triggering RNA sequence induces formation of RNA molecules containing the signal RNA sequence; and

(e) detecting the presence of said signal RNA sequence, positive results indicating the presence of said assayed nucleic acid sequence in said sample.

2. A method according to claim 1, wherein after step (b) the temperature is transiently raised to a point in which only essentially perfectly matched hybrids formed in step (b) remain hybridized while all other hybrids in which the individual strands do not perfectly match to one another are melted.

3. A method according to claim 2, wherein after raising the temperature, blocker DNA or RNA are added which are complementary to a sequence in said first or said second DNA molecule, whereby said blocker molecules compete with the assayed DNA sequence in hybridization to the first or the second DNA molecule, and consequently rehybridization of assayed DNA sequences which are not essentially perfectly complementary to the molecules of said detection ensemble is avoided.

4. The method according to claim 1, wherein said promotor sequence is double-stranded in its essential parts and hence functional.

5. The method according to claim 1, wherein said promotor sequence is single-stranded in at least an essential part thereof and during or after step (b) a sequence which is complementary to the single-stranded part of the promoter is added, rendering the promoter functional.

6. The method according to claim 5, wherein prior to addition of the sequence complementary to the single-stranded part of the promoter sequence, blocker DNA or RNA molecules are added which hybridize to free first DNA molecules in a manner so as to avoid subsequent hybridization of the sequences complementary to the single-stranded part of the promoter to said free first DNA molecule, said blocker molecule being incapable of hybridizing to said first DNA molecules which have already hybridized to said assayed DNA sequence.

7. A method according to claim 1, wherein said second DNA molecules are bound to a solid support and following step (b) there is a washing step intended for removal of DNA molecules which are unbound to the solid support.

8. The method according to claim 1, wherein:

said RNA amplification ensemble comprises;

a fourth DNA molecule comprising a promoter which is single-stranded in at least an essential part thereof and is thus inactive, and further comprises a signal DNA sequence;

said triggering RNA sequence being complementary to said single-stranded part of the promoter of the fourth DNA molecule and upon hybridization thereof to the single-stranded part of the promoter, the promoter becomes functional and in the presence of the transcription reagents, an RNA transcript is transcribed comprising a sequence transcribed from the DNA signal sequence.

11. A method according to claim 1, wherein the detection of the presence of said assayed nucleic acid in the sample is based on the detection of a specific RNA sequence.

12. A method according to claim 1, wherein the presence of the assayed nucleic acid sequence in the sample is based on the detection of an accumulation of RNA molecules following the addition of said transcription reagents.

13. A method according to claim 12, wherein the accumulation of RNA molecules is detected by light absorbance.

19. The method of claim 1, further including a blocker nucleic acid for eliminating the hybridization of a non-perfectly matched nucleic acid sequence, which is contained in an assayed sample, to another nucleic acid sequence forming part of said detection ensemble.

9. A method for detecting the presence of an assayed nucleic acid sequence in a sample, comprising the steps of:

(a) reacting the sample with a detection ensemble comprising;

a single-stranded DNA molecule having from its 3'"'"' to its 5'"'"' end three sequence portions linked to one another comprising a first arbitrary portion, a second arbitrary portion of 1-5 bases long, and a 5'"'"' end sequence which is complementary to the 5'"'"' end portion of the assayed nucleic acid sequence;

a second DNA molecule comprising a single-stranded 3'"'"' end sequence being complementary to a 3'"'"' end portion of the assayed nucleic acid sequence, and further comprising a third arbitrary portion which third arbitrary portion is double-stranded;

(b) incubating under conditions to allow hybridization of the assayed nucleic acid sequence to said single-stranded DNA molecule and to said second DNA molecule to produce DNA hybrids;

(c) raising the temperature to a point in which only essentially perfectly matched hybrids formed in step (b) remain hybridized while all other hybrids in which the individual strands do not perfectly match to one another are melted;

(d) adding a blocker DNA molecule, being a single-stranded DNA molecule comprising at its 5'"'"' end a sequence complementary to said second arbitrary portion and a sequence, at its 3'"'"' end complementary to the 5'"'"' end portion to the assayed DNA, providing conditions allowing hybridization of said blocker molecule with fee said single-stranded DNA molecule;

(e) adding a promoter DNA molecule, comprising a double-stranded functional promoter and a sequence complementary to the first and second arbitrary portions attached to the non-template strand of the promoter, and providing conditions for hybridization, whereby a hybrid comprising the assayed DNA sequence, said promoter DNA molecule, said single-stranded DNA and said second DNA molecule is obtained from which an RNA transcript can be transcribed;

(f) adding transcription reagents comprising a DNA-dependent RNA polymerase and RNA nucleotides and incubating under conditions to allow the formation of RNA transcripts having a triggering RNA sequence capable of initiating a reaction in an appropriate transcription system in which an RNA molecule having a signal RNA sequence is being produced;

(g) contacting the RNA transcripts with an RNA amplification ensemble in which the triggering RNA sequence induces formation of RNA molecules containing the signal RNA sequence; and

(h) detecting the presence of said signal RNA sequence, positive results indicating the presence of said assayed nucleic acid sequence in said sample.

10. A method according to claim 9, wherein the conditions for hybridization in step (e), are lowering of temperature.

14. A kit for use in detecting a nucleic acid sequence in a sample comprising:

a first DNA molecule comprising a double stranded promotor at its 3'"'"' end and a single stranded sequence complementary to a 5'"'"' end portion of the assayed nucleic acid sequence;

a second DNA molecule comprising a single-stranded sequence complementary to a 3'"'"' end portion of the assayed nucleic acid sequence, and further comprising a sequence which can be transcribed into a detectable RNA molecule; and

an RNA polymerase for transcribing said first and second oligonucleiotides;

wherein said detectable RNA molecule comprises (a) a self-replicating signal molecule, (b) the transcripts of said first and second DNA molecules, and (c) a stop sequence to prevent replication of the transcripts of said first and second DNA molecules.

15. The kit according to claim 14 wherein said detectable RNA molecule forms a loop.

16. The kit according to claim 15 wherein the hybridized arms of said loop form a functional promoter.

20. The kit of claim 14, further including a blocker nucleic acid for eliminating the hybridization of a non-perfectly matched nucleic acid sequence which is contained in said sample, to another nucleic acid sequence foaming part of said detection ensemble.

17. Assay components for selectively amplifying an RNA molecule in a sample, said components comprising:

a first promoter having a single stranded sequence at the 3'"'"' end of its non-coding strand adapted to hybridize to the 3'"'"' end of said RNA molecule;

a second promoter having a single stranded sequence at the 3'"'"' end of its non-coding strand adapted to hybridize to the 3'"'"' end of the transcript of said RNA molecule;

ribozymes adapted to cleave any portion of a first or second promoter which is transcribed; and

,

RNA polymerase adapted to recognize said first and second promoters and transcribe the sequences downstream thereof.

18. An assay for detecting a nucleic acid sequence in a sample comprising:

a first DNA molecule having a promoter and a 5'"'"' end complementary to the 5'"'"' end of the assayed nucleic acid sequence;

a second DNA molecule having a single-stranded 3'"'"' end complementary to the 3'"'"' end of the assayed nucleic acid sequence and 5'"'"' end which can be transcribed to an RNA molecule adapted to be selectively amplified by the assay components of claim 17; and

means for detecting said amplified RNA molecule so as to indicate the presence of said assayed nucleic acid sequence in the medium.

21. The assay components of claim 18, further including a blocker nucleic acid for eliminating the hybridization of a nor-perfectly matched nucleic acid sequence which is contained in said sample, to another nucleic acid sequence forming part of said detection ensemble.