Compositions and methods for the analysis of degraded nucleic acids

• US 20060281108A1
• Filed: 05/03/2006
• Published: 12/14/2006
• Est. Priority Date: 05/03/2005
• Status: Abandoned Application

• Current Assignee: Altheadx, Inc.
• Original Assignee: Althea Technologies Inc.
• Inventors: Ferre, Francois, Oades, Kahuku, Monforte, Joseph
• Application Number:
  US11/417,672
• Publication Number:
  US 20060281108A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/204 specific length of the oligonucleotides
• 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

Abstract

The invention relates to compositions and methods for gene expression analysis. In some embodiments, the invention provides compositions and methods for amplifying targets in a degraded nucleic acid sample. In some embodiments, the invention provides methods for determining the quality of nucleic acids (e.g., the degree of degradation) in a nucleic acid sample. The invention also provides methods for producing a gene expression profile from a degraded RNA sample.

Claims

1. A method for amplifying members of a population of degraded nucleic acids in a sample, the method comprising:

a) providing a sample comprising said population of degraded nucleic acids;

b) providing target primer pairs, wherein

i) each target primer pair comprises a forward target primer and a reverse target primer;

ii) said forward and reverse target primers each comprise (A) a target-specific nucleotide sequence that is complementary to a nucleotide subsequence of at least one member of the population of degraded nucleic acids, and (B) at least one universal priming sequence, wherein the universal priming sequence is 5′

relative to the target-specific sequence; and

c) annealing said target primer pairs to their cognate degraded nucleic acid;

d) enzymatically producing a plurality of products corresponding to subsequences of said cognate degraded nucleic acids;

e) enzymatically amplifying said plurality of products using at least one universal primer to produce a plurality of target amplicons, wherein said universal primer comprises nucleotide sequence that is complementary to said universal priming sequence, thereby amplifying members of a population of degraded nucleic acids.

2. The method of claim 1, wherein said nucleic acids are DNA, and said population of degraded nucleic acids in said sample has a mean size of not more than 1,000 nucleotides.

3. The method of claim 1, wherein said nucleic acids are RNA, and said population of degraded nucleic acids in said sample has a mean size of not more than 600 nucleotides.

4. The method of claim 1, wherein said nucleic acids are RNA, and said population of degraded nucleic acids in said sample has a mean size of about or less than 450 nucleotides.

5. The method of claim 1, wherein said nucleic acids are RNA, and said population of degraded nucleic acids in said sample has a mean size of about or less than 300 nucleotides.

6. The method of claim 1, wherein said members of a population of degraded nucleic acids comprises between about 2 and about 100 members.

7. The method of claim 1, wherein said members of a population of degraded nucleic acids comprises between about 10 and about 40 members.

8. The method of claim 1, wherein said members of a population of degraded nucleic acids correspond to expressed gene nucleotide sequences.

9. The method of claim 8, wherein at least one expressed gene nucleotide sequence is a constitutively-expressed reference gene sequence.

10. The method of claim 8, further comprising comparing a level of expression of at least one expressed gene sequence to a level of expression of at least one other expressed gene sequence.

11. The method of claim 8, wherein said expressed gene nucleotide sequences comprise tissue-specific gene sequences.

12. The method of claim 1, wherein said population of degraded nucleic acids comprises RNA or DNA.

13. The method of claim 1, wherein said population of degraded nucleic acids comprises RNA, and the step of producing a plurality of products comprises reverse transcription.

14. The method of claim 1, wherein said sample comprises mammalian RNA comprising 28S and 18S RNA species, and the quantitative ratio of 28S RNA to 18S RNA is not more than 2.0:

1.

15. The method of claim 1, wherein said sample comprises mammalian RNA comprising 28S and 18S RNA species, and the quantitative ratio of 28S RNA to 18S RNA is not more than 1.8:

1.

16. The method of claim 1, wherein said sample comprises total cellular RNA.

17. The method of claim 1, wherein said sample comprises polyadenylated RNA.

18. The method of claim 1, wherein said sample is derived from a tissue sample that has undergone fixation.

19. The method of claim 1, wherein said sample is derived from a paraffin-embedded tissue sample.

20. The method of claim 1, wherein at least one member in the plurality of products comprises not more than 200 base pairs of nucleotide sequence corresponding to said degraded nucleic acids.

21. The method of claim 1, wherein at least one member in the plurality of products comprises not more than 100 base pairs of nucleotide sequence corresponding to said degraded nucleic acids.

22. The method of claim 1, wherein at least one member in the plurality of products comprises not more than 80 base pairs of nucleotide sequence corresponding to said degraded nucleic acids.

23. The method of claim 1, wherein at least one member in the plurality of products comprises not more than 60 base pairs of nucleotide sequence corresponding to said degraded nucleic acids.

24. The method of claim 1, wherein said enzymatically producing a plurality of products comprises enzymatic nucleic acid polymerization by the polymerase chain reaction (PCR).

25. The method of claim 24, wherein said PCR is a multiplex PCR.

26. The method of claim 25, wherein said multiplex PCR employs between about 2 and about 100 target primer pairs.

27. The method of claim 25, wherein said multiplex PCR employs between about 10 and about 40 target primer pairs.

28. The method of claim 1, wherein at least one target primer further comprises at least one spacer nucleotide between said target-specific sequence and said universal priming sequence.

29. The method of claim 1, wherein the concentration of each target primer in the target primer pair is less than the concentration of said at least one universal primer.

30. The method of claim 1, wherein the ratio of the concentration of each target primer pair to the concentration of the universal primer is between about 1:

2 and 1;

1000.

31. The method of claim 1, wherein the ratio of the concentration of each target primer pair to the concentration of the universal primer is between about 1:

10 and 1;

100.

32. The method of claim 1, wherein enzymatically amplifying said plurality of products comprises enzymatic nucleic acid polymerization by the polymerase chain reaction (PCR).

33. The method of claim 1, wherein the length of one target amplicon is different than the length of at least a second target amplicon.

34. The method of claim 1, wherein each primer in a target primer pair comprises a 3′

end, and wherein said 3′

end of said forward primer is not more than 20 base pairs from said 3′

end of said reverse primer when said target primers are hybridized to a cognate nucleic acid target.

35. The method of claim 1, wherein at least one target amplicon comprises a label.

36. The method of claim 1, wherein a plurality of said target amplicons each comprise a different label.

37. The method of claim 1, comprising detecting said plurality of target amplicons.

38. The method of claim 37, wherein said detecting step is by capillary electrophoresis analysis.

39. The method of claim 37, wherein said detecting step is by hybridization analysis.

40. The method of claim 39, wherein said hybridization analysis comprises an array hybridization.

41. The method of claim 39, wherein said hybridization analysis comprises a bead system hybridization.

42. A method for determining nucleic acid quality in a nucleic acid sample, the method comprising:

a) providing a nucleic acid sample;

b) providing at least two target primer pairs, wherein;

i) each target primer pair comprises a forward target primer and a reverse target primer;

ii) said forward and reverse target primers each comprise a target-specific nucleotide sequence that is complementary to a subsequence of at least one nucleic acid in said sample; and

c) annealing said target primer pairs to their cognate nucleic acid;

d) enzymatically producing at least two products corresponding to nucleotide subsequences of said cognate nucleic acid, wherein each nucleotide subsequence is a different length;

e) enzymatically amplifying said products to produce at least two target amplicons;

f) quantitating said at least two target amplicons; and

g) comparing quantities of said target amplicons, thereby determining nucleic acid quality in said sample.

43. The method of claim 42, wherein said at least two target primer pairs each anneal to the same nucleic acid.

44. The method of claim 43, wherein said at least two products comprise overlapping nucleotide subsequences.

45. The method of claim 43, wherein said at least two products do not comprise overlapping nucleotide subsequences.

46. The method of claim 42, wherein said forward and reverse target primers each further comprise at least one universal priming sequence, wherein said universal priming sequence is 5′

relative to the target-specific sequence; and

wherein enzymatically amplifying said products comprises incorporating at least one universal primer to produce at least two target amplicons, wherein said universal primer comprises nucleotide sequence that is complementary to said universal priming sequence.

47. The method of claim 45, wherein at least one target primer further comprises at least one spacer nucleotide between said target-specific sequence and said universal priming sequence.

48. The method of claim 42, wherein said at least two target primer pairs anneal to different nucleic acids.

49. The method of claim 42, wherein each product comprises nucleotide sequence corresponding to a cognate nucleic acid, where the nucleotide sequence has a size range selected from about (i) 40-60 base pairs, inclusive;

(ii) 100-120 base pairs, inclusive; and

(iii) 180-200 base pairs, inclusive, where the nucleotide sequence size range for one product is different than the nucleotide sequence size range of any other product.

50. The method of claim 42, wherein said at least two target primer pairs consists of two target primer pairs.

51. The method of claim 42, wherein said at least two target primer pairs consists of three target primer pairs.

52. The method of claim 51, wherein each product comprises nucleotide sequence corresponding to a cognate nucleic acid, where the nucleotide sequence has a size range selected from about (i) 40-60 base pairs, inclusive;

(ii) 100-120 base pairs, inclusive; and

(iii) 180-200 base pairs, inclusive, where the nucleotide sequence size range for one product is different than the nucleotide sequence size range of the remaining two products.

53. The method of claim 42, wherein said comparing quantities of said target amplicons comprises comparing relative molar concentrations of said target amplicons.

54. The method of claim 53, wherein the relative molar concentration of one target amplicon is less than the relative molar concentration of at least a second target amplicon, thereby indicating degraded nucleic acid.

55. A method for producing a gene expression profile from a degraded RNA sample, the method comprising the steps:

a) providing a sample comprising degraded RNA, wherein said RNA corresponds to expressed genes;

b) providing a plurality of target primer pairs, wherein;

i) each target primer pair comprises a forward target primer and a reverse target primer;

ii) said forward and reverse target primers each comprise (A) a target-specific nucleotide sequence that is complementary to a nucleotide subsequence of at least one degraded RNA in said sample, and (B) at least one universal priming sequence, wherein the universal priming sequence is 5′

relative to the target-specific sequence; and

c) annealing said target primer pairs to their cognate degraded RNA;

d) enzymatically producing a plurality of products corresponding to subsequences of said cognate degraded RNA;

e) enzymatically amplifying said plurality of products using at least one universal primer to produce a plurality of target amplicons, wherein said universal primer comprises nucleotide sequence that is complementary to said universal priming sequence, thereby producing a gene expression profile from a degraded RNA sample.

56. The method of claim 55, wherein said plurality of target primer pairs comprises between about between about 2 and about 100 target primer pairs.

57. The method of claim 55, wherein said plurality of target primer pairs comprises between about between about 10 and about 40 target primer pairs.

58. The method of claim 55, wherein at least one target amplicon comprises a label.

59. The method of claim 55, wherein a plurality of said target amplicons each comprise a different label.

60. The method of claim 55, comprising detecting said plurality of target amplicons.

61. The method of claim 55, wherein said detecting step is by capillary electrophoresis analysis.

62. The method of claim 55, wherein said detecting step is by hybridization analysis.

63. The method of claim 55, wherein said hybridization analysis comprises an array hybridization.

64. The method of claim 55, wherein said hybridization analysis comprises a bead system hybridization.

65. A kit for analyzing a sample, said sample comprising degraded nucleic acid, the kit comprising:

a) a plurality of target primer pairs, wherein;

i) each target primer pair comprises a forward target primer and a reverse target primer;

ii) said forward and reverse target primers each comprise (A) a target-specific nucleotide sequence that is complementary to a nucleotide subsequence of at least one degraded nucleic acid in said sample, and (B) at least one universal priming sequence, wherein the universal priming sequence is 5′

relative to the target-specific sequence; and

iii) each primer in a target primer pair comprises a 3′

end, and wherein said 3′

end of said forward primer is not more than 20 base pairs from said 3′

end of said reverse primer when said target primers are hybridized to a cognate nucleic acid target; and

b) instructions for analyzing a sample comprising degraded nucleic acids.