Preparation of nucleic acid samples

• US 6,613,516 B1
• Filed: 10/11/2000
• Issued: 09/02/2003
• Est. Priority Date: 10/30/1999
• Status: Active Grant

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Wu, Kai, Gunderson, Kevin, Miyada, Charles G., Yang, Qing, Gingeras, Thomas, Rosenow, Carsten, Do, Duc, Christians, Fred C.
• Primary Examiner(s): ZITOMER, STEPHANIE W
• Application Number:
  US09/689,937
• Time in Patent Office: 1,056 Days
• Field of Search: 435/6, 435/287.1, 435/287.9, 435/2.1, 435/91.1, 435/196, 435/7.5, 435/7.72, 435/7.9, 435/7.92, 435/15, 435/18, 435/252.33, 435/849, 536/25.3, 536/25.4
• US Class Current: 435/5
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: Y10S 977/958 Of biomolecule property

Abstract

The presently claimed invention provides methods, compositions, and apparatus for studying nucleic acids. Specifically, the present invention provides a novel enrichment and labeling strategy for ribonucleic acids. In one embodiment, the invention provides enriching for a population of interest in a complex population by diminishing the presence of a target sequence. In a further embodiment, the invention can be used to reproducibly label and detect extremely small amounts of nucleic acids.

Claims

1. A method of preparing labeled fragments of a population of nucleic acids of interest comprising:

increasing the relative percentage of said population of nucleic acids of interest within a mixed population of nucleic acids, wherein said mixed population comprises a plurality of nucleic acid sequences, comprising;

(a) contacting a nucleic acid sample with a nucleic acid bait molecule, wherein said nucleic acid sample comprises said nucleic acids of interest and at least one unwanted target sequence and wherein said bait molecule binds specifically to said unwanted target sequence, but not to said nucleic acids of interest, under such conditions as to allow for the formation of a bait;

target complex;

(b) digesting the unwanted target sequence in the bait;

target complex thereby resulting in an increase in the relative percentage of said nucleic acids of interest within said mixed population of nucleic acids;

fragmenting said nucleic acids of interest to produce fragments; and

adding a label to the fragments.

2. The method of claim 1 wherein the nucleic acid sample is an RNA sample.

3. The method of claim 1 wherein the nucleic acid sample is derived from a prokaryotic organism.

4. The method of claim 1 wherein the nucleic acid sample is derived from a gram negative prokaryotic organism.

5. The method of claim 1 wherein the nucleic acid sample is derived from E. coli.

6. The method of claim 1 wherein said nucleic acids of interest are messenger RNA (mRNA).

7. The method of claim 1 wherein said unwanted target sequence is rRNA or tRNA.

8. The method of claim 1 wherein said target sequence is ribosomal RNA (rRNA).

9. The method of claim 1 wherein said unwanted target sequence is 23S RNA.

10. The method of claim 1 wherein said unwanted target sequence is 16S RNA.

11. The method of claim 1 wherein said bait molecule is generated from a population of nucleic acids other than the nucleic acid of interest.

12. The method of claim 1 wherein said bait molecule is chemically synthesized.

13. The method of claim 1 wherein said bait molecule is cloned from single stranded phage DNA.

14. The method of claim 1 wherein said bait molecule is synthesized by hybridizing a primer to said unwanted RNA target sequence and extending said primer by reverse transcriptase using said target sequence as a template.

15. The method of claim 1 wherein the nucleic acid sample is an RNA sample, the bait molecule is DNA, and the bait:

target complex is a DNA;

RNA hybrid.

16. The method of claim 14 wherein said bait molecules are synthesized by reverse transcriptase after the addition of at least one primer complementary to 16S RNA and at least one primer complementary to 23S RNA.

17. The method of claim 1 wherein said bait molecule is attached to a solid substrate.

18. The method of claim 17 wherein said solid substrate is a bead.

19. The method of claim 1 wherein said bait molecule is modified to comprise a selectable element.

20. The method of claim 19 wherein said selectable element is selected from the group consisting of:

a nucleic acid sequence, a ligand, a receptor, an antibody, a haptenic group, an antigen, an enzyme or an enzyme inhibitor.

21. The method of claim 19 further comprising the step of exposing said bait:

target complex to a reagent that binds said selectable element to form a reagent;

bait;

target complex prior to the step of digesting the unwanted target sequence.

22. The method of claim 21 wherein the reagent that binds said selectable element is selected from the group consisting of:

a nucleic acid sequence, a ligand, a receptor, an antibody, a haptenic group, an antigen, an enzyme or an enzyme inhibitor.

23. The method of claim 19 wherein said selectable element is a biotin.

24. The method of claim 21 wherein said selectable element is biotin and said reagent that binds said selectable element is streptavidin.

25. The method of claim 21 further comprising separating said reagent:

bait;

target complex from said mixed population prior to the step of digesting the unwanted target sequence.

26. The method of claim 25 wherein the reagent:

bait;

target complex is attached to a solid support.

27. The method of claim 15 wherein the step of digesting said unwanted target sequence comprises exposing said DNA:

RNA hybrid to a reagent which digests RNA in a DNA;

RNA hybrid and digesting said unwanted target sequence and wherein said reagent is RNAse H.

28. The method of claim 1 further comprising the step of removing the bait molecule after digesting the target sequence in said bait:

target complex.

29. The method of claim 27 further comprising the step of removing any remaining DNA bait molecules after said unwanted target sequence is removed.

30. The method of claim 29 wherein said step of removing said DNA bait molecules is accomplished by digestion with DNAse I.

31. The method of claim 28 wherein steps (a) and (b) are repeated.

32. The method of claim 31 wherein the same bait molecule is used to remove multiple target sequences.

34. The method of claim 31 wherein step (a) is performed at a first temperature and step (b) is performed at a second temperature.

35. The method of claim 1 wherein said label is a biotin.

36. The method of claim 1 wherein said label is a polyethylene oxide-Iodoacetyl Biotin.

37. The method of claim 1 wherein the label is attached to the 5′

ends of said fragments.

38. The method of claim 1 wherein after said step of fragmenting, said 5′

ends of said fragments are chemically modified.

39. The method of claim 38 wherein the 5′

ends of said fragments are chemically modified by γ

-S-ATP and T4 kinase.

40. The method of claim 38 wherein said chemical modification results in the addition of a thiol group to the 5′

end of said fragments.

41. The method of claim 40 wherein said detectable signal moiety is polyethylene oxide-lodoacetyl Biotin.

33. The method of 32 wherein a thermostable RNAse H is used to remove said target sequences from said bait:

target complex.