Method for 3′ end-labeling ribonucleic acids

• US 6,201,112 B1
• Filed: 07/22/1999
• Issued: 03/13/2001
• 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): Brusca, John S.
• Assistant Examiner(s): Lundgren, Jeffrey S.
• Application Number:
  US09/359,564
• Time in Patent Office: 600 Days
• Field of Search: 435/6, 435/68.1, 435/91.1, 435/69.1, 435/91.2, 435/193, 435/183, 536/23.1, 536/24.3
• US Class Current: 536/23.1
• 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 method of end-labeling a ribonucleic acid, said method comprising:

covalently attaching with a prokaryotic poly(A) polymerase at least one ribonucleotide to the 3′

end of said ribonucleic acid, wherein the ribonucleotide is an ATP analog labeled with a cyanine compound;

whereby said ribonucleic acid is end-labeled.

2. The method according to claim 1, wherein said covalently attaching step comprises contacting said ribonucleic acid with said labeled ribonucleotide in the presence of said prokaryotic poly(A) polymerase under conditions sufficient for said polymerase to covalently attach said labeled ribonucleotide to said 3′

end of said ribonucleic acid.

3. The method according to claim 2, wherein said prokaryotic poly(A) polymerase is a bacterial poly(A) polymerase.

4. The method according to claim 1, wherein said labeled ribonucleotide is fluorescently labeled.

8. The method according to claim 1, wherein said fluorescently labeled ribonucleotide analog is selected from the group consisting of:

CY5-ATP dyes;

CY7-ATP dyes; and

CY3-ATP dyes.

5. A method of fluorescently end-labeling a ribonucleic acid, said method comprising:

contacting said ribonucleic acid with fluorescently labeled ribonucleotide analog in the presence of a bacterial poly(A) polymerase under conditions sufficient for said polymerase to covalently attach at least one fluorescently labeled ribonucleotide analog to the 3′

end of said ribonucleic acid, wherein the fluorescently labeled ribonucleotide is an ATP analog labeled with a cyanine compound;

whereby said ribonucleic acid is fluorescently end-labeled.

6. The method according to claim 5, wherein said fluorescently labeled ribonucleotide analog comprises a fluorophore having at least two fused rings.

7. The method according to claim 6, wherein said fluorophore has a molecular weight ranging from about 200 to 2000 daltons.

9. The method according to claim 5, wherein said fluorescently labeled ribonucleotide analog is selected from the group consisting of:

CY5-ATP dyes;

CY7-ATP dyes; and

CY3-ATP dyes.