Methods for quantitative amplification and detection over a wide dynamic range

• US 8,932,817 B2
• Filed: 10/28/2013
• Issued: 01/13/2015
• Est. Priority Date: 07/21/2009
• Status: Active Grant

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: Kacian, Daniel L., Browne, Kenneth A.
• Primary Examiner(s): HORLICK, KENNETH R
• Application Number:
  US14/064,427
• Publication Number:
  US 20140072974A1
• Time in Patent Office: 442 Days
• Field of Search: 435/6.12, 435/91.2
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• 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 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2545/101 with an internal standard/control

Abstract

Disclosed are compositions and methods for making differentiable amplicon species at unequal ratios using a single amplification system in a single vessel. The number of differentiable amplicons and their ratios to one another are chosen to span the required linear dynamic range for the amplification reaction and to accommodate limitations of the measuring system used to determine the amount of amplicon generated. Unequal amounts of distinguishable amplicon species are generated by providing unequal amounts of one or more amplification reaction components (e.g., distinguishable amplification oligomers, natural and unnatural NTP in an NTP mix, or the like). The amount of target nucleic acid present in a test sample is determined using the linear detection range generated from detection of one or more amplicon species having an amount within the dynamic range of detection.

Claims

1. A method for detecting a target nucleic acid sequence in a sample comprising:

(a) providing a sample suspected of containing a target nucleic acid;

(b) generating from the target nucleic acid a defined ratio of at least two differentiable amplicon species,

wherein the at least two differentiable amplicon species are generated using a single amplification oligomer that hybridizes to one strand of the target nucleic acid and at least two amplification oligomers that hybridize to the complementary strand of the target nucleic acid,

wherein the at least two amplification oligomers hybridizing to the complementary strand of the target nucleic acid are provided in different amounts and have distinct nucleic acid sequences, such that the defined ratio of the at least two differentiable amplicon species is generated and whereby the at least two differentiable amplicon species differ in nucleic acid composition; and

(c) detecting the presence and amount of each generated amplicon species,

wherein a first amplicon species is detectable in a first linear range representing a first concentration of the target nucleic acid in the sample to a second concentration of the target nucleic acid in the sample, and a second amplicon species is detectable in a second linear range representing a third concentration of the target nucleic acid in the sample to a fourth concentration of the target nucleic acid in the sample, and

wherein the first concentration is less than the third concentration, which is less than the second concentration, which is less than the fourth concentration, such that said first and second linear ranges overlap and provide an extended dynamic range for determining the presence and amount of the target nucleic acid in the sample;

wherein the at least two amplification oligomers hybridizing to the complementary strand of the target nucleic acid hybridize to the same sequence on said strand, and one of the at least two amplification oligomers contains one or more nucleobase substitutions relative to the other;

wherein the generating and detecting steps are each performed in a single vessel; and

wherein the detecting step comprises hybridizing the at least two differentiable amplicon species with distinguishable probes that each hybridize to only one of the at least two differentiable amplicons.

2. The method of claim 1, wherein the single amplification oligomer that hybridizes to one strand of the target nucleic acid is a promoter-based amplification oligomer.

3. The method of claim 1, wherein the single amplification oligomer that hybridizes to one strand of the target nucleic acid is a primer.

4. The method of claim 1, wherein the at least two amplification oligomers that hybridize to the complementary strand of the target nucleic acid are promoter-based amplification oligomers.

5. The method of claim 1, wherein the at least two amplification oligomers that hybridize to the complementary strand of the target nucleic acid are primers.

6. The method of claim 1, wherein the number of copies of each amplicon species differs by at least three orders of magnitude.

7. The method of claim 1, wherein the number of copies of each amplicon species differs by at least four orders of magnitude.

8. The method of claim 1, wherein the amounts of the at least two amplification oligomers hybridizing to the complementary strand of the target nucleic acid differ by at least two orders of magnitude.

9. The method of claim 1, wherein the dynamic range is from about 10³ to 10⁷.

10. The method of claim 1, wherein the dynamic range is from about 10⁴ to 10⁶.

11. A method for detecting a target nucleic acid sequence in a sample comprising:

(a) providing a sample suspected of containing a target nucleic acid;

(b) generating from the target nucleic acid a defined ratio of at least two differentiable amplicon species,

wherein the at least two differentiable amplicon species are generated using a single amplification oligomer that hybridizes to one strand of the target nucleic acid and at least two amplification oligomers that hybridize to the complementary strand of the target nucleic acid,

wherein the at least two amplification oligomers hybridizing to the complementary strand of the target nucleic acid are provided in different amounts and have distinct nucleic acid sequences, such that the defined ratio of the at least two differentiable amplicon species is generated and whereby the at least two differentiable amplicon species differ in nucleic acid composition; and

(c) detecting the presence and amount of each generated amplicon species,

wherein a first amplicon species is detectable in a first linear range representing a first concentration of the target nucleic acid in the sample to a second concentration of the target nucleic acid in the sample, and a second amplicon species is detectable in a second linear range representing a third concentration of the target nucleic acid in the sample to a fourth concentration of the target nucleic acid in the sample, and

wherein the first concentration is less than the third concentration, which is less than the second concentration, which is less than the fourth concentration, such that said first and second linear ranges overlap and provide an extended dynamic range for determining the presence and amount of the target nucleic acid in the sample;

wherein the at least two amplification oligomers hybridizing to the complementary strand of the target nucleic acid comprise identical nucleotide sequences except that each of the at least two amplification oligomers comprises a unique unhybridized nucleotide sequence that is at least one nucleobase in length and that is joined to the 5′

end of the target hybridizing region of the amplification oligomer;

wherein the generating and detecting steps are each performed in a single vessel; and

wherein the detecting step comprises hybridizing the at least two differentiable amplicon species with distinguishable probes that each hybridize to only one of the at least two differentiable amplicons.

12. The method of claim 11, wherein the single amplification oligomer that hybridizes to one strand of the target nucleic acid is a promoter-based amplification oligomer.

13. The method of claim 11, wherein the single amplification oligomer that hybridizes to one strand of the target nucleic acid is a primer.

14. The method of claim 11, wherein the at least two amplification oligomers that hybridize to the complementary strand of the target nucleic acid are promoter-based amplification oligomers.

15. The method of claim 11, wherein the at least two amplification oligomers that hybridize to the complementary strand of the target nucleic acid are primers.

16. The method of claim 11, wherein the number of copies of each amplicon species differs by at least three orders of magnitude.

17. The method of claim 11, wherein the number of copies of each amplicon species differs by at least four orders of magnitude.

18. The method of claim 11, wherein the amounts of the at least two amplification oligomers hybridizing to the complementary strand of the target nucleic acid differ by at least two orders of magnitude.

19. The method of claim 11, wherein the dynamic range is from about 10³ to 10⁷.

20. The method of claim 11, wherein the dynamic range is from about 10⁴ to 10⁶.