Detection of nucleic acids through amplification of surrogate nucleic acids
View Patent ↗Methods for detecting and optionally quantitating one or more target nucleic acids are provided, in which a surrogate nucleic acid is captured to each target nucleic acid, amplified, and detected. Compositions, kit, and systems related to the methods are also described.
1. A method of detecting a first target nucleic acid, the method comprising:
providing a sample comprising the first target nucleic acid;
associating the first target nucleic acid with a solid support by
providing a first set of m target capture probes, where m is at least one, which target capture probes are capable of hybridizing to the first target nucleic acid,
hybridizing the first set of target capture probes with the first target nucleic acid, and
associating the first set of target capture probes with the solid support;
providing a first surrogate nucleic acid;
providing a first set of two or more surrogate capture probes, each of which surrogate capture probes is capable of hybridizing simultaneously to the first target nucleic acid and to a nucleic acid comprising the first surrogate nucleic acid;
hybridizing the first set of surrogate capture probes to the first target nucleic acid and to the nucleic acid comprising the first surrogate nucleic acid, thereby physically associating the first surrogate nucleic acid with the first target nucleic acid, to provide captured first surrogate nucleic acid;
amplifying the captured first surrogate nucleic acid, without amplifying the first target nucleic acid or a portion thereof, to provide amplified first surrogate nucleic acid; and
detecting the amplified first surrogate nucleic acid, whereby the presence or amount of the amplified first surrogate nucleic acid detected provides an indication of presence or amount of the first target nucleic acid in the sample;
wherein one or more copies of the nucleic acid comprising the first surrogate nucleic acid are associated with a copy of the first target nucleic acid; wherein the first set of surrogate capture probes comprises a subset of surrogate capture probes for each of the one or more copies of the nucleic acid comprising the first surrogate nucleic acid; wherein each subset of surrogate capture probes comprises n surrogate capture probes, where n is at least two; and wherein the 5′ end of each of the surrogate capture probes in the first set hybridizes to the first target nucleic acid while the 3′ end hybridizes to the nucleic acid comprising the first surrogate nucleic acid, or wherein the 3′ end of each of the surrogate capture probes in the first set hybridizes to the first target nucleic acid while the 5′ end hybridizes to the nucleic acid comprising the first surrogate nucleic acid.
2. The method of claim 1 , wherein associating the first surrogate nucleic acid with the first target nucleic acid comprises associating the first surrogate nucleic acid with the first target nucleic acid at a molar ratio of about 1:1.
3. The method of claim 1 , wherein associating the first surrogate nucleic acid with the first target nucleic acid comprises associating the first surrogate nucleic acid with the first target nucleic acid at a molar ratio of at least 2:1, at least 3:1, at least 5:1, or at least 10:1 first surrogate nucleic acid:first target nucleic acid.
4. The method of claim 1 , wherein the surrogate capture probes hybridize to nonoverlapping polynucleotide sequences in the first target nucleic acid.
5. The method of claim 1 , wherein hybridizing each subset of n surrogate capture probes to a copy of the nucleic acid comprising the first surrogate nucleic acid associates that copy with the first target nucleic acid, while hybridization of n-1 of the n surrogate capture probes to the copy of the nucleic acid comprising the first surrogate nucleic acid does not associate it with the first target nucleic acid.
6. The method of claim 1 , wherein hybridizing the first set of two or more surrogate capture probes to the first target nucleic acid and to the nucleic acid comprising the first surrogate nucleic acid is performed at a hybridization temperature which is greater than a melting temperature T m of a complex between each individual surrogate capture probe and the nucleic acid comprising the first surrogate nucleic acid.
7. The method of claim 1 , wherein m is at least two.
8. The method of claim 7 , wherein the target capture probes hybridize to nonoverlapping polynucleotide sequences in the first target nucleic acid.
9. The method of claim 1 , wherein a first support capture probe is bound to the solid support, and wherein associating the first set of target capture probes with the solid support comprises hybridizing the target capture probes of the first set with the first support capture probe.
10. The method of claim 9 , wherein m is at least two, and wherein hybridizing the first set of m target capture probes to the support capture probe captures the first target nucleic acid on the solid support, while hybridization of m-1 of the target capture probes to the support capture probe does not capture the first target nucleic acid on the solid support.
11. The method of claim 9 , wherein m is at least two, wherein hybridizing the first set of target capture probes with the first support capture probe is performed at a hybridization temperature which is greater than a melting temperature T m of a complex between each individual target capture probe and the support capture probe.
12. The method of claim 1 , wherein the solid support comprises a plurality of particles.
13. The method of claim 1 , comprising separating materials not associated with the solid support from the solid support.
14. The method of claim 1 , wherein amplifying the captured first surrogate nucleic acid to provide amplified first surrogate nucleic acid and detecting the amplified first surrogate nucleic acid comprises performing a quantitative real-time PCR experiment.
15. The method of claim 1 , wherein the first target nucleic acid is an RNA.
16. The method of claim 1 , wherein the first surrogate nucleic acid is a DNA.
17. The method of claim 1 , wherein the first target nucleic acid is derived from an animal, a human, a plant, a cultured cell, a microorganism, a virus, a bacterium, or a pathogen.
18. The method of claim 1 , wherein the sample comprises one or more of: a cell lysate, a tissue homogenate, an intercellular fluid, a bodily fluid, or a conditioned culture medium.
19. The method of claim 1 , wherein the sample is derived from one or more of: a tissue, a formalin-fixed paraffin embedded tissue, a biopsy, or a tumor.
20. The method of claim 1 , wherein the sample comprises a second target nucleic acid, the method further comprising:
providing a second surrogate nucleic acid;
physically associating the second surrogate nucleic acid with the second target nucleic acid, to provide captured second surrogate nucleic acid;
amplifying the captured second surrogate nucleic acid to provide amplified second surrogate nucleic acid; and
detecting the amplified second surrogate nucleic acid, whereby the presence or amount of the amplified second surrogate nucleic acid detected provides an indication of presence or amount of the second target nucleic acid in the sample.
21. The method of claim 20 , wherein detecting the amplified first surrogate nucleic acid and the amplified second surrogate nucleic acid comprises physically separating the amplified first surrogate nucleic acid from the amplified second surrogate nucleic acid.
22. The method of claim 1 , wherein associating the first surrogate nucleic acid with the first target nucleic acid comprises associating the first surrogate nucleic acid with the first target nucleic acid at a molar ratio of about 2:1, about 3:1, about 5:1, or about 10:1 first surrogate nucleic acid:first target nucleic acid.