Method for 3′ end-labeling ribonucleic acids

• US 6,723,509 B2
• Filed: 03/09/2001
• Issued: 04/20/2004
• Est. Priority Date: 07/22/1999
• Status: Expired due to Fees

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Ach, Robert A.
• Primary Examiner(s): BENZION, GARY
• Assistant Examiner(s): BENZION, GARY
• Application Number:
  US09/802,358
• Publication Number:
  US 20010009762A1
• Time in Patent Office: 1,138 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/24.3, 935/6, 436/518
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/68 involving nucleic acids

Abstract

Methods of end-labeling ribonucleic acids with non-radioactively labeled ribonucleotides, and particularly fluorescently labeled ribonucleotides, are provided. In the subject methods, a ribonucleic acid is contacted with a non-radioactively labeled ribonucleotide in the presence of a prokaryotic, usually bacterial, poly(A) polymerase under conditions sufficient for covalent bonding of the labeled ribonucleotide to the 3′ end of the ribonucleic acid to occur. Also provided are kits for practicing the subject method. The subject methods and kits find use in a variety of applications where labeling of the 3′ end of a ribonucleic acid with a non-radioactive label, particularly a fluorescent label, is desired.

Claims

1. A kit for use in end-labeling ribonucleic acids with non-radioactively labeled ribonucleotides, said kit comprising:

a fluorescently labeled ribonucleotide; and

a bacterial poly(A) polymerase.

2. The kit according to claim 1, wherein said fluorescently labled ribonucleotide is a fluorescently labeled ATP analog, CTP analog, UTP analog or GTP analog.

3. The kit according to claim 1, wherein said fluorescently labeled ribonucleotide contains a modified nitrogenous base moiety covalently bonded to a fluorescent label.

4. The kit according to claim 1, wherein said fluorescently labeled ribonucleotide is labeled with a xanthenic fluorophore or polymethine fluorophore.

5. The kit according to claim 4, wherein said polymethine fluorophore is a cyanine fluorophore.

6. The kit according to claim 1, wherein said fluorescently labeled ribonucleotide is labeled with a fluorophore chosen from Pyrene, Coumarin, Diethylaminocoumarin, PAM, Fluorescein Chlorotriazinyl, Fluorescein, R110, Easin, JOE, R6G, Tetramethyirhodamine, TAMRA, Lissamine, ROX, Napthofiuorescein, Texas Red, FITC, HEX, Cy3, Cy5 and Cy7.

7. The kit according to claim 1, wherein said bacterial polymerase is chosen from Escherichia coli poly(A) polymerase 1, Escherichia coli poly(A) polymerase 2, Bacillus subtilis poly(A) polymerase 1, and Bacillus subtilis poly(A) polymerase 2.

8. A kit for use in end-labeling ribonucleic acids with non-radioactively labeled ribonucleotides, said kit comprising:

a fluorescently labeled ribonucleotide labeled with a xanthenic fluorophore or cyanine fluorophore; and

a prokaryotic poly (A) polymerase.

9. The kit according to claim 8, wherein said fluorescently labeled ribonucleotide is an ATP analog, CTP analog, UTP analog or GTP analog labeled with a Xanthenic fluorophore or Cyanine fluorophore.

10. The kit according to claim 8, wherein said fluorescently labeled ribonucleotide contains a modified nitrogenous base moiety covalently bonded to said fluorophore.

11. The kit according to claim 8, wherein said fluorescently labeled ribonucleotide is labeled with a Xanthenic fluorophore chosen from FAM, Fluorescein Chlorotriazinyl, Fluorescein, JOE, R110, R6G, Tetramethylrhodaniine, TAMRA, Lissaniine, ROX, FITC, and HEX.

12. The kit according to claim 8, wherein said fluorescently labeled ribonucleotide is labeled with a cyanine fluorophore chosen from Cy3, Cy5 and Cy7.

13. The kit according to claim 8, wherein said prokaryotic poly(A) polymerase is a bacterial polymerase.

14. The kit according to claim 13, wherein said bacterial polymerase is chosen from Escherichia coli poly(A) polymerase 1, Escherichia coli poly(A) polymerase 2, Bacillus subtilis poly(A) polymerase 1, and Bacillus subtilis poly(A) polymerase 2.