Amplification and detection method

• US 7,495,093 B2
• Filed: 10/26/2007
• Issued: 02/24/2009
• Est. Priority Date: 07/16/1996
• Status: Expired due to Fees

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: Becker, Michael M., Majlessi, Mehrdad R.
• Primary Examiner(s): CALAMITA, HEATHER
• Application Number:
  US11/924,910
• Publication Number:
  US 20080090246A1
• Time in Patent Office: 487 Days
• Field of Search: None
• US Class Current: 536/24.3
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• 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 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2563/113 the label being electroactive, e.g. redox labels
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin

Abstract

The present invention concerns oligonucleotides containing one or more modified nucleotides which increase the binding affinity of the oligonucleotides to target nucleic acids having a complementary nucleotide base sequence. These modified oligonucleotides hybridize to the target sequence at a faster rate than unmodified oligonucleotides having an identical nucleotide base sequence. Such modified oligonucleotides include oligonucleotides containing at least one 2′-O-methylribofuranosyl moiety joined to a nitrogenous base. Oligonucleotides can be modified in accordance with the present invention to preferentially bind RNA targets. The present invention also concerns methods of using these modified oligonucleotides and kits containing the same.

Claims

1. A method of amplifying and detecting the presence of a target nucleic acid in a reaction mixture, the method comprising the steps of:

(a) performing a transcription-based amplification reaction to produce a transcription product having an RNA sequence contained within or complementary to a RNA or equivalent DNA sequence of the target nucleic acid; and

(b) detecting the transcription product as an indication of the presence of the target nucleic acid in the reaction mixture, the transcription product forming a hybridization complex with a target binding region of a probe molecule, wherein the target binding region comprises at least one 2′

-O-methyl ribonucleotide.

2. The method of claim 1, wherein the target binding region comprising a cluster of at least 4 contiguous 2′

-O-methyl ribonucleotides.

3. The method of claim 1, wherein the target binding region consists of 2′

-O-methyl ribonucleotides.

4. The method of claim 1, wherein the probe molecules comprises a conjugate molecule.

5. The method of claim 2, wherein the probe molecules comprises a conjugate molecule joined to the target binding region at a site located within the cluster of 2′

-O-methyl ribonucleotides.

6. The method of claim 1, wherein the probe molecules is from 10 to and 100 nucleotide bases in length.

7. The method of claim 1, wherein the probe molecules is from 10 to 16 nucleotide bases in length.

8. The method of claim 1, wherein the label is a chemiluminescent or a fluorescent agent.

9. The method of claim 1, wherein the probe molecules comprise self-complementary regions which include the at least one 2′

-O-methyl ribonucleotide.

10. The method of claim 1, wherein the transcription product comprises a structured region, and wherein the target binding region forms a hybridization complex with the structured region in step (b).

11. The method of claim 1 further comprising the step of quantifying the amount of target nucleic acid present in the reaction mixture.

12. The method of claim 1, wherein the amplification reaction is a transcription-mediated amplification reaction.