Methods and compositions for quantitative amplification and detection over a wide dynamic range

• US 8,628,924 B2
• Filed: 07/21/2010
• Issued: 01/14/2014
• 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:
  US12/840,971
• Publication Number:
  US 20110020824A1
• Time in Patent Office: 1,273 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 quantitating a target nucleic acid sequence in a test sample comprising:

(a) contacting the test sample with a nucleic acid amplification reaction mixture comprising at least two primers, wherein the at least two primers hybridize to the same strand of the target nucleic acid sequence but hybridize to distinct nucleotide sequences on the strand, and each of the at least two primers is present in the reaction mixture in a different amount, and wherein the at least two primers comprise a first inner primer and a second outer primer;

(b) subjecting the reaction mixture to amplification conditions under which each of the at least two primers simultaneously and independently produces one of at least two amplicons, wherein the number of copies of each amplicon produced differs by at least two orders of magnitude, wherein the at least two amplicons comprise a first amplicon produced from the first inner primer and a second amplicon produced from the second outer primer, and wherein the first amplicon is shorter than the second amplicon;

(c) hybridizing the at least two amplicons with at least two probes, wherein one of the at least two probes is specific to a nucleotide sequence common to both the first and second amplicons and another of the at least two probes is specific to a nucleotide sequence contained within the second amplicon but not within the first amplicon, each probe is detectable within a detection range, wherein the sum of the detection ranges for each of the at least two probes is greater than the detection range for each probe, such that the at least two probes together are detectable across a dynamic range of from about 10³ to 10⁷, and the at least two probes produce distinguishable detection signals;

(d) detecting at least one of the at least two probes; and

(e) determining the initial amount of target nucleic acid sequence in the test sample.

2. The method of claim 1, wherein the reaction mixture further comprises a promoter-primer which operates in a direction opposite the first inner and second outer primers.

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

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

5. The method of claim 1, wherein the amount of each primer differs by at least two orders of magnitude.

6. The method of claim 1, wherein the at least two probes are acridinium ester probes.

7. The method of claim 1, wherein the at least two probes are HICS probes.

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

9. A method for quantitating a nucleic acid in a test sample comprising:

(a) providing a test sample suspected of containing a target nucleic acid in an amount T₁;

(b) contacting the test sample with a nucleic acid amplification reaction mixture comprising at least two primers, wherein the at least two primers hybridize to the same strand of the target nucleic acid sequence but each primer hybridizes to a distinct nucleotide sequence on the strand, and wherein each primer is present in the reaction mixture in a different amount P_X, wherein each P_X differs by at least two orders of magnitude, and where x is an integer between 1 and the number of primers in the mixture, and wherein the at least two primers comprise a first inner primer and a second outer primer;

(c) subjecting the reaction mixture to amplification conditions under which each of the at least two primers simultaneously and independently produces one of at least two amplicons, wherein for each amplicon, a number of copies A_x is produced, wherein each A_x differs by at least two orders of magnitude, wherein the at least two amplicons comprise a first amplicon produced from the first inner primer and a second amplicon produced from the second outer primer, and wherein the first amplicon is shorter than the second amplicon;

(d) hybridizing the at least two amplicons with at least two probes, wherein one of the at least two probes is specific to a nucleotide sequence common to both the first and second amplicons and another of the at least two probes is specific to a nucleotide sequence contained within the second amplicon but not within the first amplicon, each probe is detectable within a detection range C_x(a) to C_x(b), wherein C_x(a) is the minimum detectable number of copies of amplicon and C_x(b) is the maximum detectable number of copies of amplicon for probe x, wherein for each probe, C_x+1(a) is greater than C_x(a) and C_x+1(b) is greater than C_X(b) such that the at least two probes together are detectable across a dynamic range of from about 10³ to 10⁷, wherein for each amplicon-probe combination, A_x is between C_x(a) and C_x(b), and wherein the at least two probes produce distinguishable detection signals;

(e) detecting at least one of the at least two probes; and

(f) determining the initial amount of target nucleic acid sequence in the test sample.

10. The method of claim 9, wherein the reaction mixture further comprises a promoter-primer which operates in a direction opposite the first inner and second outer primers.

11. The method of claim 9, wherein the number of copies of each amplicon produced differs by at least three orders of magnitude.

12. The method of claim 9, wherein the number of copies of each amplicon produced differs by at least four orders of magnitude.

13. The method of claim 9, wherein the at least two probes are acridinium ester probes.

14. The method of claim 9, wherein the at least two probes are HICS probes.

15. The method of claim 9, wherein the dynamic range is from about 10⁴ to 10⁶.