Passive internal references for the detection of nucleic acid amplification products

• US 5,736,333 A
• Filed: 06/04/1996
• Issued: 04/07/1998
• Est. Priority Date: 06/04/1996
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Perkinelmer Incorporated
• Inventors: Livak, Kenneth J., McBride, Lincoln J.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US08/657,989
• Time in Patent Office: 672 Days
• Field of Search: 435/6, 435/91.2, 435/810, 536/23.1
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 2525/173 incorporating a polynucleotide run, e.g. polyAs, polyTs
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

The invention relates to passive internal references for use in quantitating the formation of amplification products in a nucleic amplification reaction. The internal amplification reference molecules of the invention comprise a first and second fluorophore joined together through a backbone connector. The first and second fluorophores are joined on the backbone in a configuration that permits the energy transfer from the first fluorophore to the second fluorophore. The backbone connector is selected so as not to bind to the target nucleic acid sequence under nucleic acid amplification conditions. Preferably, the backbone connector is a polynucleotide. Another aspect of the invention is to provide passive internal reference molecule containing reagent compositions for use in nucleic acid amplification reactions. The compositions comprise the internal amplification reference molecule of the invention and a nucleic acid amplification reaction buffer. The reagent compositions, optionally, include additional components required for nucleic acid amplification reactions. The invention also provides improved methods of measuring the amount of amplification product in nucleic acid amplification reactions employing fluorescer-quencher probe assays, including methods for the real-time measurement of amplification product formation. The methods comprise the step of adding the internal reference molecule of the invention to the amplification reaction mixture. Fluorescence of the second fluorophore on the internal reference may then be measured and used to calculate changes in fluorescence of the fluorophore on a fluorescer-quencher probe.

Claims

1. A reagent composition, said composition comprising:

a nucleic acid amplification buffer, and

an internal reference molecule, said internal reference molecule comprising

a first fluorophore,

a second fluorophore, and

a backbone connector that does not hybridize in a sequence specific manner to a polynucleotide for amplification under nucleic acid amplification conditions, wherein the backbone connector joins the first and second fluorophores so as to permit the transfer of energy from the first fluorophore to the second fluorophore.

2. A composition according to claim 1, wherein the backbone connector is a polynucleotide.

3. A composition according to claim 2, wherein the polynucleotide is 2 to 25 nucleotides in length.

4. A composition according to claim 3, wherein the polynucleotide is (dT)8.

5. A composition according to claim 1 wherein the first fluorophore is selected from the group consisting of fluorescein, 6-carboxyfluorescein, 2'"'"',4'"'"',5'"'"',7'"'"',-tetrachloro-4,7-dichlorofluorescein, 2'"'"',7'"'"'-dimethoxy-4'"'"',5'"'"'-6-carboxyrhodamine (JOE), N'"'"',N'"'"',N'"'"',N'"'"'-tetramethyl-6-carboxyrhodamine (TAMRA) and 6-carboxy-X-rhodamine (ROX).

6. A composition according to claim 5, wherein the first fluorophore is 6-carboxyfluorescein.

7. A composition according to claim 5 wherein the second fluorophore is selected from the group consisting of fluorescein, 6-carboxyfluorescein, 2'"'"',4'"'"',5'"'"',7'"'"',-tetrachloro-4,7-dichlorofluorescein, 2'"'"',7'"'"'-dimethoxy-4'"'"',5'"'"'-6-carboxyrhodamine (JOE), N'"'"',N'"'"',N'"'"',N'"'"'-tetramethyl-6-carboxyrhodamine (TAMRA) and 6-carboxy-X-rhodamine (ROX).

8. A composition according to claim 6, wherein the second fluorophore is 6-carboxy-X-rhodamine.

18. A kit for performing nucleic acid amplification reactions, said kit comprising a reagent composition according to claim 1 and one or more reagents selected from the group consisting of a fluorescer-quencher probe and a thermostable DNA polymerase.

19. A kit according to claim 18, wherein said reagent is a fluorescer-quencher probe.

20. A kit according to claim 18, wherein said reagent is a thermostable DNA polymerase.

21. A kit according to claim 20, said kit further comprising a fluorescer-quencher probe.

9. In a method of measuring the amount of an amplification product of a polynucleotide for amplification in a nucleic acid reaction wherein the improvement comprises,

adding an internal amplification reference molecule to the amplification reaction, said internal reference molecule comprising

a first fluorophore,

a second fluorophore, and

a backbone connector that does not hybridize to the polynucleotide for amplification under nucleic acid amplification conditions,

wherein the backbone connector joins the first and second fluorophores so as to permit the transfer of energy from the first fluorophore to the second fluorophore.

10. A method according to claim 9, wherein the nucleic acid amplification reaction is measured in real-time.

11. A method according to claim 9, wherein the backbone connector is a polynucleotide.

12. A method according to claim 10, wherein the polynucleotide is 2 to 25 nucleotides in length.

13. A method according to claim 12, wherein the polynucleotide is (dT)8.

14. A method according to claim 9 wherein the first fluorophore is selected from the group consisting of fluorescein, 6-carboxyfluorescein, 2'"'"',4'"'"',5'"'"',7'"'"',-tetrachloro-4,7-dichlorofluorescein, 2'"'"',7'"'"'-dimethoxy-4'"'"',5'"'"'-6-carboxyrhodamine (JOE), N'"'"',N'"'"',N'"'"',N'"'"'-tetramethyl-6-carboxyrhodamine (TAMRA) and 6-carboxy-X-rhodamine (ROX).

15. A method according to claim 14, wherein the first fluorophore is 6-carboxyfluorescein.

16. A method according to claim 14 wherein the second fluorophore is selected from the group consisting of fluorescein, 6-carboxyfluorescein, 2'"'"',4'"'"',5'"'"',7'"'"',-tetrachloro-4,7-dichlorofluorescein, 2'"'"',7'"'"'-dimethoxy-4'"'"',5'"'"'-6-carboxyrhodamine (JOE), N'"'"',N'"'"',N'"'"',N'"'"'-tetramethyl-6-carboxyrhodamine (TAMRA) and 6-carboxy-X-rhodamine (ROX).

17. A method according to claim 15, wherein the second fluorophore is 6-carboxy-X-rhodamine.