Use of blocker oligonucleotides in selective amplification of target sequences

• US 8,574,847 B2
• Filed: 09/23/2011
• Issued: 11/05/2013
• Est. Priority Date: 04/27/2009
• Status: Active Grant

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: Becker, Michael M., Livezey, Kristin W.
• Primary Examiner(s): HORLICK, KENNETH R
• Application Number:
  US13/242,895
• Publication Number:
  US 20120009629A1
• Time in Patent Office: 774 Days
• Field of Search: 435/6.12, 435/91.2
• US Class Current: 435/6.12
• 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 1/6865 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 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2531/143 Promoter based amplification, e.g. NASBA, 3SR, TAS

Abstract

A method for the selective amplification of a target sequence. The method includes hybridizing a tagged oligonucleotide to the target sequence and reducing the effective concentration of unhybridized tagged oligonucleotide which is capable of hybridizing to the target sequence. The tagged oligonucleotide includes a target hybridizing sequence and a tag sequence situated 5′ to target hybridizing sequence, where the tag sequence does not stably hybridize to a target nucleic acid containing the target sequence. A blocker oligonucleotide is provided which is designed to hybridize to a region of a nucleic acid containing the target sequence, where the region targeted by the blocker oligonucleotide is 3′ to the target sequence. Amplification products are formed using first and second oligonucleotides. The first oligonucleotide hybridizes to the 3′-end of the complement of the target sequence, and the second oligonucleotide hybridizes to the complement of the tag sequence.

Claims

1. A method for the selective amplification of at least one target nucleic acid sequence from a sample, the method comprising the ordered steps of:

(a) treating a sample comprising a target nucleic acid with a tagged oligonucleotide comprising first and second regions, the first region comprising a target hybridizing sequence which hybridizes to a 3′

-end of a target nucleic acid sequence contained in the target nucleic acid and the second region comprising a tag sequence situated 5′

to the target hybridizing sequence, wherein the second region does not stably hybridize to the target nucleic acid;

(b) reducing in the sample the effective concentration of unhybridized tagged oligonucleotide having an active form in which a target hybridizing sequence of the unhybridized tagged oligonucleotide is available for hybridization to the target nucleic acid;

(c) providing a blocker oligonucleotide to the sample under conditions permitting the blocker oligonucleotide to stably hybridize to a target region of a contaminating nucleic acid that is introduced into the sample after step (b), wherein the contaminating nucleic acid contains the target nucleic acid sequence, wherein the target region is situated 3′

to and spaced no more than 10 bases from the 3′

-end of the target nucleic acid sequence contained in the contaminating nucleic acid, and wherein the blocker oligonucleotide is modified to prevent the initiation of DNA synthesis therefrom; and

(d) producing amplification products in a nucleic acid amplification reaction using first and second oligonucleotides, wherein the first oligonucleotide comprises a hybridizing sequence which hybridizes to a 3′

-end of the complement of the target nucleic acid sequence and the second oligonucleotide comprises a hybridizing sequence which hybridizes to the complement of the tag sequence, wherein the second oligonucleotide does stably hybridize to the target nucleic acid, and wherein each of the amplification products comprises a base sequence which is substantially identical or complementary to the base sequence of the target nucleic acid sequence and further comprises a base sequence which is substantially identical or complementary to all or a portion of the tag sequence.

2. The method of claim 1, wherein step (b) comprises removing unhybridized tagged oligonucleotide from the sample.

3. The method of claim 2, wherein the target nucleic acid is immobilized on a solid support during step (b).

4. The method of claim 1, wherein step (b) does not include the use of an enzyme having a nuclease activity.

5. The method of claim 1, wherein the tagged oligonucleotide has an active form during step (a) which permits the target hybridizing sequence to hybridize to the target nucleic acid sequence, and wherein all or a portion of the unhybridized tagged oligonucleotide is converted to an inactive form in step (b) which blocks or prevents the tagged oligonucleotide from hybridizing to the target nucleic acid sequence during step (c).

6. The method of claim 5, wherein step (a) is performed under a first set of conditions, step (b) is performed under a second set of conditions, and step (c) is performed under a third set of conditions, and wherein the conditions of steps (b) and (c) are less stringent than the conditions of step (a).

7. The method of claim 5, wherein the temperature of the sample is lowered between steps (a) and (b).

8. The method of claim 5, wherein all or a portion of the unhybridized tagged oligonucleotide from step (a) is converted from a single-stranded form to a duplexed form in step (b).

9. The method of claim 8, wherein the duplexed form is a hairpin tag molecule comprising a tag closing sequence joined to a 5′

-end of the tagged oligonucleotide, wherein the tag closing sequence hybridizes to the target hybridizing sequence under the conditions of step (b), thereby blocking hybridization of unhybridized tagged oligonucleotide from step (a) to the target nucleic acid sequence in steps (b) and (c).

10. The method of claim 9, wherein the tagged oligonucleotide further comprises a third region containing a promoter for an RNA polymerase, the third region being situated 5′

to the second region.

11. The method of claim 9, wherein the tag closing sequence is joined to the tagged oligonucleotide by a non-nucleotide linker.

12. The method of claim 8, wherein the target hybridizing sequence of unhybridized tagged oligonucleotide is hybridized to a tag closing oligonucleotide in step (b), the tagged oligonucleotide and the tag closing oligonucleotide being distinct molecules.

13. The method of claim 12, wherein the tagged oligonucleotide and the tag closing oligonucleotide are both present in the sample during step (a), and wherein the target hybridizing sequence favors hybridization to the target nucleic acid sequence over the tag closing oligonucleotide in step (a).

14. The method of claim 12, wherein the tag closing oligonucleotide is modified to prevent the initiation of DNA synthesis therefrom.

15. The method of claim 1, wherein the blocker oligonucleotide is from 12 to 24 bases in length.

16. The method of claim 1, wherein the blocker oligonculeotide includes nucleotide analogs exhibiting high affinity for the target region and which prevent the initiation of DNA synthesis therefrom.

17. The method of claim 16, wherein the nucleotide analogs are LNA or 2′

-O-methyl ribonucleotides.

18. The method of claim 1, wherein the conditions of the nucleic acid amplification reaction are isothermal.

19. The method of claim 1, wherein the nucleic acid sample is exposed to a known contaminating source of the target nucleic acid sequence after step (b), and wherein the production of the amplification products is substantially limited to amplification of target nucleic acid sequence contributed by the nucleic acid sample and not by the contaminating source of the target nucleic acid sequence.