AMPLIFICATION AND DETECTION METHOD

• US 20080090246A1
• Filed: 10/26/2007
• Published: 04/17/2008
• Est. Priority Date: 07/16/1996
• Status: Active Grant

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: Brentano, Steven, MAJLESSI, Mehrdad, BECKER, Michael
• Granted Patent: US 7,495,093 B2
• Time in Patent Office: Days
• US Class Current: 435/6.18
• 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:

performing a transcription-based amplification reaction to produce transcription products having a sequence contained within or complementary to a sequence of the target nucleic acid; and

detecting the transcription products during the amplification reaction as an indication of the presence of the target nucleic acid in the reaction mixture, wherein the transcription products complex with a region of probe molecules comprising at least one 2′

-O-methyl ribonucleotide.

2. The method of claim 1, wherein the region of the probe molecules which complexes with the transcription products includes a cluster of at least 4 contiguous 2′

-O-methyl ribonucleotides.

3. The method of claim 1, wherein the region of the probe molecules which complexes with the transcription products consists of 2′

-O-methyl ribonucleotides.

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

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

-O-methyl ribonucleotides.

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

7. The method of claim 1, wherein the probe molecules are 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 probe molecules complex with structured regions of the transcription products under the conditions of the amplification reaction.

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

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