Methods for the enrichment of low-abundance polynucleotides

• US 20040014105A1
• Filed: 05/09/2003
• Published: 01/22/2004
• Est. Priority Date: 05/09/2002
• Status: Abandoned Application

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Schroth, Gary P., Schroeder, Benjamin G., Chen, Caifu
• Application Number:
  US10/435,489
• Publication Number:
  US 20040014105A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/107 incorporating a peptide nucleic acid
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety

Abstract

The invention relates to methods for the selective enrichment of low-abundance polynucleotides in a sample. These methods use enzymatically non-extendable nucleobase oligomers to selectively block polymerase activity on high abundance species, thereby resulting in an enrichment of less abundant species in the sample. The invention also relates to the pools of enriched polynucleotides produced by the methods. The resulting pools of enriched polynucleotides find a variety of uses, including the analysis of gene expression and the creation of cDNA libraries.

Claims

1. A method for the enrichment of a low abundance polynucleotide relative to a high abundance polynucleotide in a sample, wherein the ratio of the high abundance polynucleotide to the low abundance polynucleotide is at least about 10:

1, the method comprising

(a) exposing said sample to at least one first enzymatically non-extendable nucleobase oligomer having a nucleobase sequence complementary to a sequence within the high abundance polynucleotide under conditions such that base pairing occurs;

(b) exposing said sample to a primer having a nucleobase sequence complementary to a sequence within the low abundance polynucleotide under conditions such that base pairing occurs; and

(c) subjecting said sample to conditions for polymerase extension, such that said low abundance polynucleotide is amplified by extension of the primer and the high abundance polynucleotide is not amplified.

2. The method of claim 1, wherein the ratio of the high abundance polynucleotide to the low abundance polynucleotide is at least 100:

1.

3. The method of claim 1, wherein the sample comprises at a first and a second high abundance polynucleotide and in step (a) is exposed to at least two enzymatically non-extendable nucleobase oligomers, wherein one nucleobase oligomer comprises a nucleobase sequence that is complementary to a sequence within the first high abundance polynucleotides and the second nucleobase oligomer comprises a nucleobase sequence that is complementary to a sequence within the second high abundance polynucleotide.

4. The method of claim 1, wherein the low abundance polynucleotide and high abundance polynucleotide are RNA molecules selected from the group consisting of mRNA, rRNA, cRNA and tRNA molecules.

5. The method of claim 1, wherein the low abundance and high abundance polynucleotides are cDNA molecules.

6. The method of claim 1, wherein said enzymatically non-extendable nucleobase oligomer does not have a ribose-containing oligomeric structure.

7. The method of claim 6, wherein said enzymatically non-extendable nucleobase oligomer is a peptide nucleic acid (PNA) oligomer.

8. The method of claim 1, wherein said enzymatically non-extendabale nucleobase oligomer is a modified nucleotide oligomer or internucleotide analog oligomer.

9. The method of claim 8, wherein said modified nucleotide oligomer is selected from the group consisting of 2′

-modified and 3′

-modified nucleotide oligomers.

10. The method of claim 9, wherein said 2′

-modified and 3′

-modified nucleotide oligomer is selected from the group consisting of 2′

-O-alkyl modified nucleotide oligomers and 3′

-alkyl modified nucleotide oligomers.

11. The method of claim 10, wherein said 2′

-O-alkyl modified nucleotide oligomers are 2′

-o-methyl nucleotide oligomers.

12. The method of claim 8, wherein said modified nucleotide oligomer or internucleotide analog oligomer is selected from locked nucleic acids (LNA), N3′

-P5′

phosphoramidate (NP) oligomers, minor groove binder-linked-oligonucleotides (MGB-linked oligonucleotides), phosphorothioate (PS) oligomers, C₁-C₄ alkylphosphonate oligomers, phosphoramidates, β

-phosphodiester oligonucleotides, and a-phosphodiester oligonucleotides.

13. The method of claim 12, wherein said C₁-C₄ alkylphosphonate oligomers are methyl phosphonate (MP) oligomers.

14. The method of claim 1, wherein said enzymatically non-extendable first nucleobase oligomer is chimeric.

15. The method of claim 1, wherein said sample comprises more than one high abundance polynucleotide.

16. The method of claim 1, wherein said sample of polynucleotides comprises polynucleotides selected from the group consisting of RNA and DNA.

17. The method of claim 1, wherein said sample of polynucleotides comprises RNA, and polymerase extension is by reverse transcription to yield a first strand cDNA.

18. The method of claim 17, wherein said method further comprises second strand cDNA synthesis.

19. The method of claim 18, wherein said sample is exposed to at least one enzymatically non-extendable nucleobase oligomer during first strand cDNA synthesis.

20. The method of claim 18, wherein said sample is exposed to at least one enzymatically non-extendable nucleobase oligomer during second strand cDNA synthesis.

21. The method of claim 18, wherein said sample is exposed to at least one enzymatically non-extendable nucleobase oligomer during both first strand cDNA synthesis and second strand cDNA synthesis.

22. The method of claim 18, wherein said method further comprises an amplification step.

23. The method of claim 22 wherein said amplification step is by polymerase chain reaction.

24. The method of claim 22 wherein said amplification step is by in vitro transcription.

25. The method of claim 16 wherein said RNA is mRNA or cRNA or total cellular RNA.

26. The method of claim 1 wherein said sample of polynucleotides comprises DNA, and polymerase extension is by DNA-dependent DNA-polymerase in a polymerase chain reaction.

27. The method of claim 22, further comprising a step of labeling said amplified polynucleotides.

28. The method of claim 27, wherein said labeling is concomitant with amplification.

29. The method of claim 27, wherein said labeling is subsequent to amplification.

30. A plurality of polynucleotides, where the relative abundance of at least one target polynucleotide has been reduced relative to a non-target polynucleotide, and wherein at least one target polynucleotide is selected from the list of genes recited in FIG. 14.

31. The plurality of polynucleotides of claim 30, where the relative abundance of at least one non-target polynucleotide has been increased relative to a target polynucleotide.

32. The plurality of polynucleotides of claim 30, where the plurality of polynucleotides are DNA molecules or RNA molecules.

33. The plurality of polynucleotides of claim 32, where the DNA molecules are cDNA molecules.

34. The plurality of polynucleotides of claim 32, where the RNA molecules are cRNA molecules.

35. The plurality of polynucleotides of claim 30, where the polynucleotides are labeled.

36. The plurality of polynucleotides of claim 33, where the cDNA molecules are cloned into a vector.

37. A kit for the enrichment of at least one low abundance polynucleotide in a sample of polynucleotides, wherein said sample comprises at least one high abundance polynucleotide and at least one low abundance polynucleotide, wherein said kit comprises at least one enzymatically non-extendable nucleobase oligomer having a nucleobase sequence complementary to said at least one high abundance target polynucleotide.

38. The kit of claim 37, wherein the sample comprises at least 5 high abundance polynucleotides and the kit comprises at least five non-enzymatically non-extendable nucleobase oligomers each having a nucleobase sequence complementary to one of the five high abundance target polynucleotides.

39. The kit of claim 37, additionally comprising a primer for amplifying the at least one low abundance polynucleotide.

40. The method of claim 39, wherein the primer is a random primer.

41. The kit of claim 37, wherein said high abundance target polynucleotide is selected from the genes recited in FIG. 14.

42. The kit of claim 37, wherein said non-extendable nucleobase oligomer is selected from peptide nucleic acid (PNA) oligomers, 2′

-O-alkyl modified nucleotide oligomers, 3′

-alkyl modified nucleotide oligomers, locked nucleic acids (LNA), N3′

-P5′

phosphoramidate (NP) oligomers, minor groove binder-linked-oligonucleotides (MGB-linked oligonucleotides), phosphorothioate (PS) oligomers, C₁-C₄ alkylphosphonate oligomers, phosphoramidates, 13-phosphodiester oligonucleotides, and a-phosphodiester oligonucleotides.

43. The kit of claim 37, further comprising one or more components selected from the group consisting of an RNA-dependent DNA polymerase (reverse transcriptase), a DNA-dependent RNA polymerase, a DNA-dependent DNA polymerase, an oligo-dT polymerase primer, an oligo-dT polymerase primer further comprising nucleotide sequence for RNA polymerase initiation, deoxyribonucleotide triphosphates, ribonucleotide triphosphates, a DNA polymerase primer suitable for cDNA second strand synthesis, and a means for polynucleotide labeling.

44. A method for analyzing gene expression in a sample having at least one high abundance polynucleotide, comprising:

(a) exposing said sample to at least one enzymatically non-extendable nucleobase oligomer having a nucleobase sequence complementary to a sequence within said high abundance polynucleotide under conditions such that base pairing occurs,

(b) subjecting said sample to conditions for polymerase extension to produce an enriched polynucleotide sample,

(c) labeling said polynucleotides in said enriched polynucleotide sample,

(d) contacting said labeled polynucleotide sample with a probe using a hybridization means to form a hybridization complex, and

(e) detecting said hybridization complex, where the detection of a hybridization complex is indicative of gene expression.

45. A method for the synthesis of a cDNA library enriched for at least one low abundance polynucleotide, comprising the steps of:

(a) providing a sample of mRNA, where said mRNA has at least one high abundance transcript and at least one low abundance transcript,

(b) exposing said sample to at least one enzymatically non-extendable nucleobase oligomer having a nucleobase sequence complementary to a sequence within said high abundance mRNA under conditions such that base pairing occurs,

(c) subjecting said sample to conditions for reverse transcription and first strand cDNA synthesis,

(d) subjecting said sample to conditions for second strand cDNA synthesis to form double stranded cDNA molecules,

(e) cloning said double stranded cDNA molecules into a vector to yield an enriched cDNA library.

46. A method of enriching a sample for one or more low abundance polynucleotides comprising:

amplifying the low abundance polynucleotides using polymerase extension while blocking amplification of at least one high abundance polynucleotide,

wherein blocking amplification of the high abundance polynucleotide comprises contacting the high abundance polynucleotide prior to amplification with an enzymatically non-extendable oligomer comprising a sequence that is complementary to a sequence within the high abundance polynucleotide under conditions such that base pairing occurs, and

wherein the ratio of the high abundance polynucleotide to each low abundance polynucleotide is at least about 10;

1.

47. The method of claim 46 wherein the sample is enriched for at least 10 low abundance polynucleotides.

48. The method of claim 46 wherein the sample is enriched for at least 100 low abundance nucleotides.

49. The method of claim 46 wherein amplification of at least 2 high abundance polypeptides is blocked.

50. The method of claim 46 wherein amplification of at least 10 high abundance polypeptides is blocked.

51. The method of claim 46 wherein amplification of at least 50 high abundance polypeptides is blocked.

52. The method of claim 46 wherein the sample is enriched for at least 10 low abundance polynucleotides and the amplification of at least 2 high abundance polypeptides is blocked.