Detection of nucleic acids through amplification of surrogate nucleic acids

• US 20070161020A1
• Filed: 11/09/2006
• Published: 07/12/2007
• Est. Priority Date: 11/10/2005
• Status: Active Grant

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Panomics, Inc.
• Inventors: Luo, Yuling, Bui, Son
• Granted Patent: US 8,017,360 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• 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 2561/113 Real time assay

Abstract

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.

Claims

1. A method of detecting a first target nucleic acid, the method comprising:

providing a sample comprising the first target nucleic acid;

providing a first surrogate nucleic acid;

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 to provide amplified first surrogate nucleic acid; and

detecting the amplified first surrogate nucleic acid, whereby 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.

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 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 method of claim 1, wherein associating the first surrogate nucleic acid with the first target nucleic acid comprises:

providing a first set of one 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; and

hybridizing the first set of one or more surrogate capture probes to the first target nucleic acid and to the nucleic acid comprising the first surrogate nucleic acid, whereby the first surrogate nucleic acid is physically associated with the first target nucleic acid.

5. The method of claim 4, wherein the first set of surrogate capture probes comprises two or more surrogate capture probes.

6. The method of claim 5, wherein the surrogate capture probes hybridize to nonoverlapping polynucleotide sequences in the first target nucleic acid.

7. The method of claim 4, 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.

8. The method of claim 7, 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.

9. The method of claim 7, wherein hybridizing the first set of one 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.

10. The method of claim 1, comprising associating the first target nucleic acid with a solid support.

11. The method of claim 10, wherein associating the first target nucleic acid with the solid support comprises:

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;

whereby the first target nucleic acid is associated with the solid support.

12. The method of claim 11, wherein m is at least two.

13. The method of claim 11, wherein the target capture probes hybridize to nonoverlapping polynucleotide sequences in the first target nucleic acid.

14. The method of claim 11, 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.

15. The method of claim 14, 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.

16. The method of claim 14, 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.

17. The method of claim 10, wherein the solid support comprises a plurality of particles.

18. The method of claim 10, comprising separating materials not associated with the solid support from the solid support.

19. 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.

20. The method of claim 1, wherein the first target nucleic acid is an RNA.

21. The method of claim 1, wherein the first surrogate nucleic acid is a DNA.

22. 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.

23. 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.

24. 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.

25. 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 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.

26. The method of claim 25, 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.

27. A composition comprising:

a first target nucleic acid;

a nucleic acid comprising a first surrogate nucleic acid;

a first set of one 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; and

one or more primers for amplifying the first surrogate nucleic acid.

28. The composition of claim 27, 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.

29. The composition of claim 27, 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.

30. The composition of claim 27, 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.

31. The composition of claim 27, wherein the first set of surrogate capture probes comprises two or more surrogate capture probes.

32. The composition of claim 31, wherein the surrogate capture probes hybridize to nonoverlapping polynucleotide sequences in the first target nucleic acid.

33. The composition of claim 27, comprising a solid support.

34. The composition of claim 33, wherein the solid support comprises a plurality of particles.

35. The composition of claim 33, comprising 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.

36. The composition of claim 35, wherein the target capture probes hybridize to nonoverlapping polynucleotide sequences in the first target nucleic acid.

37. The composition of claim 35, wherein m is at least two.

38. The composition of claim 35, 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.

39. The composition of claim 27, comprising amplified first surrogate nucleic acid.

40. The composition of claim 39, comprising one or more reagents for detecting the amplified first surrogate nucleic acid.

41. The composition of claim 27, wherein the first target nucleic acid is an RNA.

42. The composition of claim 27, wherein the first surrogate nucleic acid is a DNA.

43. The composition of claim 27, 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.

44. The composition of claim 27, further comprising:

a second nucleic acid comprising a second surrogate nucleic acid; and

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.

45. The composition of claim 44, comprising the second target nucleic acid and/or one or more primers for amplifying the second surrogate nucleic acid.

46. A kit comprising:

a nucleic acid comprising a first surrogate nucleic acid;

a first set of one 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;

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.

47. The kit of claim 46, wherein the solid support comprises a plurality of particles.

48. The kit of claim 46, comprising one or more reagents for detecting an amplified first surrogate nucleic acid.

49. The kit of claim 46, 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.