Heterogeneous assay for pyrophosphate detection

• US 6,232,075 B1
• Filed: 12/13/1999
• Issued: 05/15/2001
• Est. Priority Date: 12/14/1998
• Status: Expired due to Term

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Li & S Company Limited (Yesco Holdings Co., Ltd.)
• Inventors: Williams, John G. K.
• Primary Examiner(s): Fredman, Jeffrey
• Assistant Examiner(s): CHAKRABARTI, ARUN K
• Application Number:
  US09/460,304
• Time in Patent Office: 519 Days
• Field of Search: 435/6, 435/7, 424/12, 436/501, 935/77, 935/78
• US Class Current: 435/6.12
• CPC Class Codes: C07D 209/12 Radicals substituted by oxygen atoms
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Nucleotide triphosphate probes containing a fluorophore attached to the β-phosphate and a quencher moiety sufficiently proximal to the fluorophore moiety for use in pyrophosphate detection assays are disclosed. These probes exhibit distinguishable fluorescence characteristics when the fluorophore is attached to the nucleotide through the γ-phosphate and when it is unattached to the nucleotide. The present invention also provides kits and integrated systems for practicing the assays described herein.

Claims

1. A heterogeneous assay method for detecting pyrophosphate cleavage, the components of the assay comprising a labeled NTP, a target nucleic acid, a primer nucleic acid and a polymerase, said method comprising:

(a) flowing said labeled nucleotide triphosphate (NTP) consisting of a γ

-phosphate with a fluorophore moiety attached thereto and a quencher moiety sufficiently proximal to said fluorophore moiety to prevent fluorescence of said fluorophore moiety past an immobilized component selected from the group consisting of said polymerase and said target nucleic acid;

(b) incorporating said NTP on a primer strand hybridized to said target nucleic acid using said polymerase and releasing said γ

-phosphate with said fluorophore moiety attached thereto; and

(c) detecting said fluorescent moiety thereby detecting pyrophosphate cleavage.

2. The method according to claim 1, wherein said nucleotide triphosphate (NTP) is a member selected from the group consisting of deoxyadenosine triphosphate, deoxycytosine triphosphate, deoxyguanosine triphosphate and deoxythymidine triphosphate.

3. The method according to claim 1, wherein said nucleotide triphosphate (NTP) is a member selected from the group consisting of adenosine triphosphate, cytosine triphosphate, guanosine triphosphate and uridine triphosphate.

4. The method according to claim 1, wherein said fluorophore moiety and said quencher moiety interact via a mechanism selected from the group consisting of fluorescence resonance energy transfer, an electron transfer quenching mechanism and a ground-state complex quenching mechanism.

5. The method according to claim 1, wherein each of said plurality of fluorescent species is detected based upon a change in either intensity measurement or fluorescent lifetime measurement.

6. The method according to claim 1, wherein said nucleotide triphosphate (NTP) is a plurality of nucleotide triphosphates (NTPs).

7. The method according to claim 1, wherein each of said plurality of nucleotide triphosphates (NTPs) has an indicator of identity.

8. The method according to claim 1, wherein said polymerase is a member selected from the group consisting of a DNA polymerase, a DNA dependent RNA polymerase and a reverse transcriptase.

9. The method according to claim 8, wherein said polymerase is a DNA polymerase.

10. The method according to claim 1, wherein said polymerase is immobilized on a solid support.

11. The method according to claim 10, wherein said solid support is a member selected from the group consisting of controlled pore glass, a glass plate, polystyrene, an avidin coated polystyrene bead, cellulose, nylon, acrylamide gel and activated dextran.

12. A nucleotide triphosphate (NTP) probe, said NTP probe consisting of:

a NTP having a γ

-phosphate with a fluorophore moiety attached thereto;

a quencher moiety sufficiently proximal to said fluorophore moiety to prevent fluorescence of said fluorophore moiety;

wherein said fluorophore moiety exists quenched with at least about a 5 fold quenching efficiency when said γ

-phosphate is attached to said NTP and unquenched when said γ

-phosphate is detached from said NTP.

13. The NTP probe according to claim 12, wherein said quencher moiety is covalently bound to the base of said NTP.

14. The NTP probe according to claim 13, wherein said NTP is a member selected from the group consisting of a deoxynucleotide triphosphate (dNTP), and a nucleotide triphosphate (NTP).

15. The NTP probe according to claim 14, wherein said NTP is a deoxynucleotide triphosphate (dNTP).

16. The NTP probe according to claim 15, wherein said deoxynucleotide triphosphate (dNTP) is a member selected from the group consisting of deoxyadenosine triphosphate, deoxycytosine triphosphate, deoxyguanosine triphosphate and deoxythymidine triphosphate.

17. The NTP probe according to claim 15, wherein said nucleotide triphosphate (NTP) is a member selected from the group consisting of adenosine triphosphate, cytosine triphosphate, guanosine triphosphate and uridine triphosphate.

18. The NTP probe according to claim 13, wherein and said quencher moiety is a member selected from the group consisting of DABCYL, rhodamine, tetramethyl rhodamine, pyrene butyrate, eosine nitrotyrosine, ethidium, fluorescein, Malachite green, Texas Red, dinitrobenzene and trinitrobenzene.

19. The NTP probe according to claim 13, wherein said fluorophore moiety is a member selected from the group consisting of fluorescein, 5-carboxyfluorescein (FAM), rhodamine, 5-(2′

-aminoethyl)aminonapthalene-1-sulfonic acid (EDANS), anthranilamide, coumarin, terbium chelate derivatives, Reactive Red 4, BODIPY dyes and cyanine dyes.

20. The NTP probe according to claim 12, wherein said fluorophore moiety is attached to said γ

-phosphate via a linker.

21. The dNTP probe according to claim 20, wherein said fluorophore linker is an alkylene group having between about 5 to about 12 carbons.

22. The NTP probe according to claim 12, wherein said quencher moiety is attached to said NTP via a linker moiety.

23. The NTP probe according to claim 22, wherein said quencher moiety is attached to said NTP via an alkynylamino linker.

24. The NTP probe according to claim 22, wherein said quencher moiety is attached to said NTP via an alkynylamino linker wherein said linker is attached to the 5-position of a pyrimidine nucleotide and the 7 position of the purine nucleotide.

25. The NTP probe according to claim 22, wherein said quencher moiety is attached to said fluorophore moiety via a linker.

26. The NTP probe according to claim 25, wherein said fluorophore moiety is a fluorescein dye and said quencher moiety is a rhodamine dye.

27. The NTP probe according to claim 12, wherein said NTP probe is DABCYL-dUTP-BODIPY TR.

28. The NTP probe according to claim 12, wherein said NTP probe is DNP-dUTP-BODIPY TR.

29. A kit for assaying pyrophosphate cleavage, said kit comprising:

(a) a plurality of NTPs each cosisting of a γ

-phosphate with a distinguishing fluorophore moiety attached thereto and each having a quencher moiety sufficiently proximal to said distinguishing fluorophore moiety to prevent fluorescence of said distinguishing fluorophore moiety;

wherein said distinguishing fluorophore moiety exists quenched with at least about a 5 fold quenching efficiency when said γ

-phosphate is attached to each of said plurality of dNTP moieties and each is unquenched when said γ

-phosphate is detached from each of said plurality of dNTP moieties; and

(b) a polymerase.

30. The kit according to claim 29, wherein each of said distinguishing fluorophore moieties interacts with said quencher moiety via a mechanism which is a member selected from the group consisting of fluorescence resonance energy transfer (FRET), electron transfer and ground-state complex mechanism.