DNA/RNA target and signal amplification

• US 5,573,914 A
• Filed: 05/15/1995
• Issued: 11/12/1996
• Est. Priority Date: 09/10/1991
• Status: Expired due to Fees

• Current Assignee: GEN Trak, Inc.
• Original Assignee: GEN Trak, Inc.
• Inventors: Love, Jack D.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Campbell, Eggerton
• Application Number:
  US08/441,678
• Time in Patent Office: 547 Days
• Field of Search: 435/5, 435/6, 435/91.2, 435/91.21, 935/77, 935/78
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• 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 2521/119 RNA polymerase
• CPC Class Codes: C12Q 2525/121 incorporating both deoxyribonucleotides and ribonucleotides
• CPC Class Codes: C12Q 2525/143 incorporating a promoter sequence
• CPC Class Codes: C12Q 2531/143 Promoter based amplification, e.g. NASBA, 3SR, TAS
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: G01N 33/6854 Immunoglobulins

Abstract

A process for detecting the presence or absence of a specific nucleic acid sequence or antibody in a sample using an oligonucleotide to bind to the nucleic acid sequence or antibody to be detected, forming double-stranded nucleic acid sequence using the bound oligonucleotide in conjunction with another oligonucleotide or DNA synthesis, synthesizing RNA transcripts from the thus-formed double-stranded nucleic acid sequence, and detecting the existence of the RNA transcripts, and oligonucleotides and kits useful in carrying out such a process.

Claims

1. A process for detecting the presence or absence of at least one specific nucleic acid in a sample containing a nucleic acid or mixture of nucleic acids, which process comprises:

(a) immobilizing said specific nucleic acid to be detected,

(b) treating the sample with a first oligonucleotide under hybridizing conditions such that a portion of said first oligonucleotide hybridizes to the 3'"'"' end of said specific nucleic acid to be detected and a portion of said first oligonucleotide does not hybridize to said specific nucleic acid to be detected, wherein said portion of said first oligonucleotide which does not hybridize to said specific nucleic acid to be detected forms a double-stranded RNA polymerase binding site and a nucleotide sequence transcribing RNA when hybridized to a complementary nucleotide sequence in the absence of nucleic acid extension,

(c) treating the sample to remove any of the first oligonucleotide which is not hybridized to said specific nucleic acid to be detected,

(d) treating the sample with a second oligonucleotide under hybridizing conditions such that said second oligonucleotide hybridizes to said portion of said first oligonucleotide which does not hybridize to said specific nucleic acid to be detected and forms a double-stranded RNA polymerase binding site and nucleotide sequence transcribing RNA,

(e) treating the sample with RNA polymerase and nucleotide triphosphates such that said nucleotide sequence formed between a portion of said first oligonucleotide and a portion of said second oligonucleotide results in the synthesis of RNA transcripts from the nucleotide sequence transcribing RNA wherein the sequence of the nucleic acid to be detected is not transcribed, and

(f) determining whether said RNA transcripts were synthesized.

2. The process of claim 1, wherein the sample at step (e) is further treated with said first oligonucleotide and with DNA polymerase such that said first oligonucleotide hybridizes to said RNA transcripts which act as primers for synthesis of an extension nucleotide sequence complementary to said first oligonucleotide to form a double-stranded RNA polymerase binding site and a nucleotide sequence transcribing RNA which results in the synthesis of additional RNA transcripts.

3. The process of claim 2, wherein the step of treating the sample with said first oligonucleotide sequence and with DNA polymerase is repeated.

4. The process of claim 1, wherein said RNA transcripts are virus fragments and are replicated through use of an RNA virus replicase.

5. The process of claim 1, wherein the sample at step (c) is further treated to separate said first oligonucleotide sequence from said specific nucleic acid sequence to be detected.

6. The process of claim 5, wherein said first oligonucleotide sequence is separated from said specific nucleic acid sequence to be detected by denaturation.

7. The process of claim 1, wherein the sample at step (d) is further treated to separate said first oligonucleotide from said specific nucleic acid to be detected resulting in a soluble portion of said first oligonucleotide hybridized to said second oligonucleotide sequence forming a double-stranded RNA polymerase binding site and a nucleotide sequence transcribing RNA.

8. The process of claim 7, wherein said first oligonucleotide sequence is separated from said specific nucleic acid sequence to be detected through use of a restriction enzyme which cleaves the portion of said first oligonucleotide sequence hybridized to said specific nucleic acid sequence to be detected.

9. The process of claim 1, wherein said portion of said first oligonucleotide sequence which hybridizes to said second oligonucleotide sequence includes a nucleotide sequence non-identical and a nucleotide sequence identical to said portion of said first oligonucleotide sequence which hybridizes to said specific nucleic acid sequence to be detected.

10. The process of claim 9, wherein said portion of said first oligonucleotide sequence which hybridizes to said second oligonucleotide sequence is identical to said portion of said first oligonucleotide sequence which hybridizes to said specific nucleic acid sequence to be detected.

11. A process for detecting the presence or absence of at least one specific nucleic acid in a sample containing a nucleic acid or mixture of nucleic acids, which process comprises:

(a) immobilizing said specific nucleic acid to be detected,

(b) treating the sample with an oligonucleotide under hybridizing conditions such that a portion of said oligonucleotide hybridizes to the 3'"'"' end of said specific nucleic acid to be detected which acts as a primer for said oligonucleotide and a portion of said oligonucleotide does not hybridize to said specific nucleic acid to be detected, wherein said portion of said oligonucleotide which does not hybridize to said specific nucleic acid to be detected forms an RNA polymerase binding site and a nucleotide sequence transcribing RNA when an extension product is synthesized from said primer, wherein said extension product is complementary to said portion of the oligonucleotide that does not hybridize to the specific nucleic acid to be detected,

(c) treating the sample with DNA polymerase, RNA polymerase, and nucleotide triphosphates such that an extension product is synthesized from said primer which is complementary to said oligonucleotide to form a double-stranded RNA polymerase binding site and a nucleotide sequence transcribing RNA which results in the synthesis of RNA transcripts from the nucleotide sequence transcribing RNA, wherein the sequence of the nucleic acid to be detected is not transcribed, and

(d) treating the RNA transcripts with said oligonucleotide and DNA polymerase such that the oligonucleotide hybridizes to the RNA transcripts, which act as primers for synthesis of an extension nucleotide sequence complementary to the oligonucleotide, to form a double-stranded RNA polymerase binding site and a nucleotide sequence transcribing RNA, resulting in the synthesis of additional RNA transcripts from the nucleotide sequence transcribing RNA, wherein the sequence of the nucleic acid to be detected is not transcribed, and

(e) determining whether said RNA transcripts were synthesized.

12. The process of claim 11 wherein the step of treating the sample with said oligonucleotide sequence and with DNA polymerase is repeated.

13. The process of claim 11 wherein said RNA transcripts are virus fragments and are replicated through use of an RNA virus replicase.