Methods of manipulating nucleic acids

• US 20060040283A1
• Filed: 04/11/2005
• Published: 02/23/2006
• Est. Priority Date: 04/11/2001
• Status: Abandoned Application

• Current Assignee: United States Department Of Health And Human Services
• Original Assignee: United States Department Of Health And Human Services
• Inventors: Brownstein, Michael J., Xiang, Charlie
• Application Number:
  US11/104,737
• Publication Number:
  US 20060040283A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6841 In situ hybridisation

Abstract

Methods are provided for labeling nucleic acid molecules for use in hybridization reactions, and kits employing these methods. The level of labeling is increased by including one or more reactive modifications, such as amine-modifications, into the primers used to initiate synthesis of the nucleic acid molecule, for instance through random-primed reverse transcription. Also provided are modified random primers (such as amine-modified random primers) useful in these methods, labeling and hybridization kits comprising such primers, labeled nucleic acid molecules and mixtures of molecules, and methods for using them. Methods are also provided for amplifying a nucleic acid template contained within extremely small samples, in some cases as little as one cell. In particular embodiments, a single random primer is used for all steps of the amplification method. The nucleic acid template can either be of cellular or viral origin. The disclosure also provides an improved method of fixing cells, tissue sections, or laser microdissected sections from which RNA can be obtained for subsequent use as RNA templates or for generating labeled probe.

Claims

1. A method of producing a modified nucleic acid probe, comprising:

contacting a nucleic acid template with a modified random primer under conditions sufficient to permit base-specific hybridization between the template and the primer, wherein the modified random oligonucleotide primer comprises an amine-modified dNTP or a label-substituted dNTP; and

polymerizing a nucleic acid molecule complementary to a nucleic acid sequence in the template and incorporating at least one modified oligonucleotide primer, thereby producing the modified nucleic acid probe.

2. The method of claim 1, wherein the modified random primer is modified at the five prime end of the primer.

3. The method of claim 1, wherein the modified random primer comprises an amine-modified dNTP, the method further comprising:

coupling the modified nucleic acid probe to a label molecule to form a label-probe conjugate.

4. A modified random primer for use in the method of claim 1.

5. The modified primer of claim 4, wherein the primer is any one of SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9, or SEQ ID NO;

10.

6. The method of claim 1, wherein the nucleic acid template comprises a mixture of nucleic acid molecules.

7. The method of claim 6, wherein the mixture of nucleic acid molecules comprises RNA.

8. The method of claim 9, wherein polymerizing comprises polymerizing a cDNA.

9. A method of producing a fluorescent hybridization probe, comprising:

contacting a template nucleic acid sample with the modified random primer of claim 4, wherein the modified random primer comprises at least one aminoallyl dUTP residue;

polymerizing a nucleic acid molecule complementary to a sequence in the template sample and incorporating one or more modified random primers, to produce a modified complementary nucleotide; and

contacting the modified complementary nucleotide with an amine-reactive fluorescent label, thereby producing the fluorescent hybridization probe.

10. The method of claim 9, wherein the template nucleic acid comprises mRNA and polymerizing comprises reverse transcription.

11. A fluorescent hybridization probe produced by the method of claim 9.

14. The method of claim 1, wherein the nucleic acid template is (A) an amplified nucleic acid template;

(B) originally isolated from a small number of cells;

or (C) both (A) and (B).

15. A kit for producing a labeled hybridization probe or for probing an array, comprising the modified random primer of claim 4.

16. The method of claim 14, wherein the small number of cells is lysed by sonication in a buffer comprising first strand buffer and an RNase inhibitor.

17. The method of claim 14, wherein the small number of cells is less than about 1000 cells, less than about 100 cells, about 10 cells, or about 1 cell.

18. The method of claim 14, wherein the amplified template comprises RNA.

19. The method of claim 18, further comprising contacting the amplified template with a second primer, wherein the second primer has a nucleic acid sequence as set forth in SEQ ID NO:

12, under conditions sufficient to permit base-specific hybridization between the template and the second primer.

20. The method of claim 19, wherein the second primer, comprising a nucleic acid sequence as set forth in SEQ ID NO:

12, is used in at least one round of cDNA synthesis other than the first round.

21. The method of claim 19, wherein the modified random primer comprises an amine-modified dNTP, the method further comprises coupling the amine-modified nucleic acid probe to a label molecule to form a label-probe conjugate.

25. The method of claim 1, wherein a second primer, comprising a nucleic acid sequence as set forth in SEQ ID NO:

12, contacts the nucleic acid template under conditions sufficient to permit base-specific hybridization between the template and the second primer and generates an amplified nucleic acid template that is capable of hybridizing with the modified random primer.

26. The method of claim 25, wherein the nucleic acid template (A) comprises a mixture of nucleic acid molecules;

(B) is isolated from a small number of cells;

or (C) is both (A) and (B).

27. The method of claim 26, wherein the mixture of nucleic acid molecules comprises RNA or DNA.

28. The method of claim 27, wherein the RNA comprises ribosomal RNA, messenger RNA, transfer RNA, or mixtures thereof.

29. The method of claim 26, wherein the template is derived from a cell or a virus.

30. The method of claim 26, wherein the small number of cells is less than about 1000 cells, less than about 100 cells, about 10 cells or is 1 cell.

31. The method of claim 26, wherein the small number of cells are infected with a virus, wherein the virus is a DNA virus or an RNA virus.

32. The method of claim 31, wherein the virus is human herpes virus-8.

33. The method of claim 34, wherein the second primer comprising a nucleic acid sequence as set forth in SEQ ID NO:

12 is used in at least one round of cDNA synthesis.

34. The method of claim 25, wherein the modified random primer is modified at the five prime end of the primer.

35. The method of claim 25, further comprising coupling the modified nucleic acid probe to a label molecule to form a label-probe conjugate.

36. The method of claim 25, wherein the modified random primer is any one of SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9, or SEQ ID NO;

10.

12. An improved method for random primer reverse transcription labeling of a nucleic acid hybridization probe, the improvement comprising using random primers modified with at least one amine-substituted dNTP or fluorescent-dye modified dNTP in the reverse transcription reaction.

13. An improved hybridization probe as produced by the method of claim 12.

22. A method of producing an RNA template from a small number of cells, comprising:

lysing a small number of cells by sonication in a buffer, wherein the buffer comprises first strand buffer and an RNase inhibitor, to produce a lysate, wherein the lysate comprises the RNA nucleic acid template.

23. The method of claim 22, wherein the small number of cells comprises less than about ten cells or about 1 cell.

24. A method of producing a modified nucleic acid probe, comprising:

amplifying the RNA template of claim 22 to produce an amplified template;

generating cDNA from the amplified template;

contacting the cDNA with a modified random primer comprising an amine-modified dNTP under conditions sufficient to permit hybridization between the cDNA and the modified random primer; and

polymerizing a nucleic acid molecule complementary to a nucleic acid sequence in the cDNA and incorporating at least one modified oligonucleotide primer, thereby producing the modified nucleic acid probe.

37. A method of amplifying a nucleic acid template, comprising:

contacting a nucleic acid template with a primer under conditions sufficient to permit base-specific hybridization between the template and the primer, wherein the primer comprises a T3-promoter and a random primer and wherein the random primer comprises between about 4 and about 12 nucleotides;

polymerizing a nucleic acid molecule complementary to a nucleic acid sequence in the template, to produce a polymerized nucleic acid molecule; and

amplifying the polymerized nucleic acid molecule, thereby amplifying a nucleic acid template.

38. The method of claim 37, wherein the primer comprises a T3N9 primer with a sequence as set forth in SEQ ID NO:

12.

39. The method of claim 37, further comprising coupling the amplified nucleic acid template to a label molecule.

40. The method of claim 39, wherein the label molecule is a fluorophore or a hapten.

41. The method of claim 39, wherein labeling the amplified nucleic acid template comprises contacting the amplified nucleic acid template with a modified random primer comprising at least one aminoallyl dNTP residue;

polymerizing a nucleic acid molecule complementary to a sequence in the amplified nucleic acid template and incorporating one or more modified random primers, to produce a modified complementary nucleotide; and

contacting the modified complementary nucleotide with an amine-reactive fluorescent label, thereby producing the labeled amplified nucleic acid template.

46. The method of claim 37, wherein the amount of primer per reaction available for base-specific hybridization was (A) less than 100 picomoles or (B) less than 10 picomoles.

42. A method of fixing a cell in order to preserve cell structure but permit extraction of high quality RNA for subsequent study, comprising:

contacting the cell with dithio-bis(Succinimidyl Propionate);

contacting the cell with a reducing agent; and

extracting RNA, thereby fixing a cell to preserve cell structure and permitting extraction of high quality RNA for subsequent study.

43. The method of claim 42, wherein the cell is a cultured cell, a cell in a tissue section, a cell in a laser capture microdissection section, or a cell in a tissue biopsy sample.

44. The method of claim 42, further comprising producing a modified nucleic acid probe, wherein the method further comprises:

contacting the extracted RNA with a modified random primer under conditions sufficient to permit base-specific hybridization between the RNA and the primer, wherein the modified random oligonucleotide primer comprises an amine-modified dNTP or a label-substituted dNTP; and

polymerizing a nucleic acid molecule complementary to a nucleic acid sequence in the template and incorporating at least one modified oligonucleotide primer, thereby producing the modified nucleic acid probe.

45. The method of claim 44, wherein the extracted RNA is amplified prior to contacting the RNA with the modified random primer.