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 composition comprising:
a first target nucleic acid;
a nucleic acid comprising a first surrogate nucleic acid;
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 the nucleic acid comprising the first surrogate nucleic acid, 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;
a solid support;
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; and
one or more primers for amplifying the first surrogate nucleic acid.
2. The composition of claim 1 , wherein the nucleic acid comprising the first surrogate nucleic acid is hybridized to the first set of surrogate capture probes, which surrogate capture probes are hybridized to the first target nucleic acid, whereby the first surrogate nucleic acid is physically associated with the first target nucleic acid at a molar ratio of about 1:1.
3. The composition of claim 1 , wherein the nucleic acid comprising the first surrogate nucleic acid is hybridized to the first set of surrogate capture probes, which surrogate capture probes are hybridized to the first target nucleic acid, whereby the first surrogate nucleic acid is physically associated with the first target nucleic acid at a molar ratio of at least about 2:1, at least about 3:1, at least about 5:1, or at least about 10:1 first surrogate nucleic acid:first target nucleic acid.
4. The composition of claim 1 , 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; and wherein each subset of surrogate capture probes comprises n surrogate capture probes, where n is at least two.
5. The composition of claim 1 , wherein the surrogate capture probes hybridize to nonoverlapping polynucleotide sequences in the first target nucleic acid.
6. The composition of claim 1 , wherein the solid support comprises a plurality of particles.
7. The composition of claim 1 , wherein the target capture probes hybridize to nonoverlapping polynucleotide sequences in the first target nucleic acid.
8. The composition of claim 1 , wherein m is at least two.
9. The composition of claim 1 , wherein the solid support comprises a first support capture probe bound to the solid support, and wherein the target capture probes of the first set are capable of hybridizing simultaneously to the first target nucleic acid and to the first support capture probe.
10. The composition of claim 1 , comprising amplified first surrogate nucleic acid.
11. The composition of claim 10 , comprising one or more reagents for detecting the amplified first surrogate nucleic acid.
12. The composition of claim 1 , wherein the first surrogate nucleic acid is a DNA.
13. The composition of claim 1 , comprising a second nucleic acid comprising a second surrogate nucleic acid; a second set of one or more surrogate capture probes, each of which surrogate capture probes is capable of hybridizing simultaneously to a second target nucleic acid and to the second nucleic acid comprising the second surrogate nucleic acid; and the second target nucleic acid and/or one or more primers for amplifying the second surrogate nucleic acid.
14. A kit comprising:
a nucleic acid comprising a first surrogate nucleic acid;
a first set of two or more surrogate capture probes, each of which surrogate capture probes is capable of hybridizing simultaneously to a first target nucleic acid and to the nucleic acid comprising the first surrogate nucleic acid, 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;
a solid support comprising a first support capture probe bound to the solid support;
a first set of m target capture probes, where m is at least one, which target capture probes are capable of hybridizing simultaneously to the first target nucleic acid and to the first support capture probe; and
one or more primers for amplifying the first surrogate nucleic acid;
packaged in one or more containers.
15. The kit of claim 14 , further comprising:
a second nucleic acid comprising a second surrogate nucleic acid;
a second set of one or more surrogate capture probes, each of which surrogate capture probes is capable of hybridizing simultaneously to a second target nucleic acid and to the second nucleic acid comprising the second surrogate nucleic acid; and
a second set of m target capture probes, where m is at least one, which target capture probes are capable of hybridizing simultaneously to the second target nucleic acid and to the first support capture probe.
16. The composition of claim 1 , wherein the first surrogate nucleic acid is physically associated with the first target nucleic acid.