Modified random primers for probe labeling

• US 20050221304A1
• Filed: 04/11/2002
• Published: 10/06/2005
• Est. Priority Date: 04/11/2001
• Status: Abandoned Application

• Current Assignee: The United States of America As Represented By The Secretary of Agriculture, United States As Represented By The Secretary Department of Health and Human Services
• Original Assignee: The United States of America As Represented By The Secretary of Agriculture
• Inventors: Brownstein, Michael J, Xiang, Charlie
• Application Number:
  US10/474,611
• Publication Number:
  US 20050221304A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences

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.

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 modified random primer of claim 4, wherein the primer is P2 (SEQ ID NO:

1).

7. The modified random primer of claim 4, wherein the primer is P4 (SEQ ID NO:

2).

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

9. The method of claim 8, wherein the mixture of nucleic acid molecules comprises RNA.

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

17. The method of claim 1, wherein the nucleic acid template is an amplified nucleic acid template.

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

19. The method of claim 1, wherein the nucleic acid template is originally isolated from a small number of cells.

20. The method of claim 19, wherein the small number of cells is lysed by sonication in a buffer comprising first strand buffer and an RNase inhibitor.

21. The method of claim 19, wherein the small number of cells is less than about 1000 cells.

22. The method of claim 19, wherein the small number of cells is less than about 100 cells.

23. The method of claim 19, wherein the small number of cells is about 10 cells.

24. The method of claim 19, wherein the small number of cells is about 1 cell.

25. The method of claim 19, wherein the nucleic acid template is an amplified template.

26. The method of claim 25, wherein the amplified template comprises RNA.

27. The method of claim 26, 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.

28. The method of claim 27, 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.

29. The method of claim 27, 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.

11. A method of producing a fluorescent hybridization probe, comprising

contacting a template nucleic acid sample with a modified random primer comprising 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.

12. The method of claim 11, wherein aminoallyl dNTP is included during polymerizing.

13. The method of claim 11, wherein the template nucleic acid comprises mRNA and polymerizing comprises reverse transcription.

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

15. 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.

16. An improved hybridization probe as produced by the method of claim 15.

30. 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.

31. The method of claim 30, wherein the small number of cells comprises less than about ten cells.

32. The method of claim 30, wherein the small number of cells comprises about one cell.

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

amplifying the RNA template of claim 30 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.