Methods and compositions for quantitative amplification and detection over a wide dynamic range
View Patent ↗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.
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 10 3 to 10 7 , 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 10 4 to 10 6 .
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 T 1 ;
(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 10 3 to 10 7 , 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 10 4 to 10 6 .