ASSAY METHODS FOR NUCLEIC ACID IN A SAMPLE

• US 20010014446A1
• Filed: 02/13/1998
• Published: 08/16/2001
• Est. Priority Date: 02/13/1998
• Status: Active Grant

• Current Assignee: BioVeris Corporation (Roche Holding AG)
• Original Assignee: BioVeris Corporation (Roche Holding AG)
• Inventors: HEROUX, JEFFREY A., RAO, SAVITHA M., CORCORAN, MARTA V.
• Granted Patent: US 6,635,418 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2545/113 with an external standard/control, i.e. control reaction is separated from the test/target reaction
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin

Abstract

The present invention relates to a method for the determination of the presence and amount of DNA in a sample. The method is based on the use of a nucleic acid template dependent enzyme in combination with a random primer to generate an enzymatic product which incorporates a binding species and a detectable species covalently linked.

Claims

1. A method for the detection and/or quantitation of nucleic acid in a sample, which comprises:

a) mixing at least one random primer at least 4 nucleotides in length, having at least one detectable species with a sample nucleic acid,

b) adding at least one NTP having at least one binding species and optionally at least one NTP,

c) adding at least one nucleic acid polymerase,

d) incubating the mixture of step c), under conditions which allow said at least one nucleic acid polymerase to be active,

e) contacting the mixture of step d) with at least one solid phase,

f) detecting and/or quantitating the amount of nucleic acid in said sample by detecting and/or quantitating the amount of said at least one detectable species bound to said solid phase.

6. A method as in

claim 1

,

2

, 3, 4, or 5 wherein said at least one binding species is selected from the group consisting of biotin, avidin, streptavidin, antibody, antigen, lectin, receptor, ligand, hormone, nucleic acid sequence, mimitope and nucleic acid base pairing polymer.

7. A method as in

claim 1

,

2

, 3, 4, or 5 wherein said at least one detectable species is selected from the group consisting of biotin, avidin, streptavidin, antibody, antigen, lectin, receptor, ligand, hormone, nucleic acid sequence, mimitope, nucleic acid base pairing polymer, fluorescent molecule, electrochemiluminescent molecule, radioactive molecule, colored molecule, peroxidase, alkaline phosphatase and enzymes capable of producing a detectable species.

8. A method as in

claim 1

,

2

, 3 or 5 wherein said at least one nucleic acid polymerase is selected from the group consisting of Taq DNA polymerase, T4 DNA polymerase, Klenow fragment (3′

-5′

), Pfu DNA polymerase, Exo- Pfu DNA polymerase, E. coli DNA polymerase I, Klenow fragment of DNA polymerase I, MMLV reverse transcriptase and AMV reverse transcriptase.

9. A method as in

claim 1

,

2

, 3, 4, or 5 wherein said at least one solid phase is selected from the group consisting of fiber, fibril, plastic surface, plastic bead, magnetic bead, plastic tube, gold surface, metal surface, metal bead and colloids.

10. A method as in

claim 1

,

2

, 3, 4, or 5 wherein said at least one NTP is selected from the group consisting of dATP, dGTP, dCTP, dUTP, dTTP, ATP, CTP, GTP, TTP, UTP inosineTP, propyne dCTP, propyne dUTP, 5-bromo dCTP, 5-iodo dUTP, 5-fluoro dUTP, O-6 methyl dGTP, 7-deaza dGTP, N-6 methyl-2′

-dATP, biotin-dATP, biotin-dCTP, biotin-dUTP, digoxigenin dUTP, digoxigenin UTP and biotin ddUTP.

11. A method as in

claim 1

,

2

, 3, 4, or 5 wherein said at least one random primer is 4-70 nucleotides.

12. A method as in

claim 1

,

2

, 3, 4, or 5 wherein said conditions comprise a solution with a pH between 5.5 and 9.5, a NTP concentration between 1 pM and 10 mM, a Mg2+ concentration between 0.05 mM and 500 mM, and a reducing agent concentration between 0 and 500 mM, where the sum of the molarities is between 1 mM and 500 mM.

14. A method as in

claim 1

wherein said random primer is from 4 to 20 nucleotides in length.

15. A method as in

claim 14

wherein said at least one label is selected from the group consisting of biotin, nucleic acid sequence, nucleic acid base pairing linear polymer, fluorescent molecule, electrochemiluminescent molecule, radioactive molecule, peroxidase and alkaline phosphatase.

16. A method as in

claim 14

wherein said at least one binding species is selected from the group consisting of biotin, antigen, lectin, ligand, hormone, nucleic acid sequence, mimitope and nucleic acid base pairing linear polymer.

17. A method as in

claim 14

wherein said at least one nucleic acid polymerase is selected from the group consisting of Taq DNA polymerase, Klenow fragment (3′

-5′

) E. coli DNA polymerase I and Klenow fragment of DNA polymerase I.

18. A method as in

claim 14

wherein said at least one solid phase is selected from the group consisting of magnetic bead, plastic plate and polymer bead.

19. A method as in

claim 14

wherein said at least one NTP is selected from the group consisting of dATP, dGTP, dCTP, dUTP, dTTP, 7-deaza dGTP, biotin-dATP, biotin-dCTP, biotin-dUTP, digoxigenin dUTP, digoxigenin UTP and biotin ddUTP.

20. A method as in

claim 14

wherein said random primer is 6-10 nucleotides in length.

21. A method as in

claim 14

wherein said conditions comprise those optimal for Klenow fragment of DNA polymerase I to synthesize DNA.

22. A method as in

claim 20

wherein said NTP is a dNTP.

2. A method for the detection and/or quantitation of nucleic acid in a sample, which comprises:

a) mixing at least one random primer at least 4 nucleotides in length, having at least one binding species with a sample nucleic acid,

b) adding at least one NTP having at least one detectable species and optionally at least one NTP,

c) adding at least one nucleic acid polymerase,

d) incubating the mixture of step c), under conditions which allow said at least one nucleic acid polymerase to be active,

e) contacting the mixture of step d) with at least one solid phase,

f) detecting and/or quantitating the amount of nucleic acid in said sample by detecting and/or quantitating the amount of said at least one detectable species bound to said solid phase.

3. A method for the detection and/or quantitation of nucleic acid in a sample, which comprises:

a) mixing at least one random primer at least 4 nucleotides in length with a sample nucleic acid,

b) adding at least one NTP having at least one binding species and optionally at least one NTP having at least one detectable species and optionally at least one NTP,

c) adding at least one nucleic acid polymerase,

d) incubating the mixture of step c), under conditions which allow said at least one nucleic acid polymerase to be active,

e) contacting the mixture of step d) with at least one solid phase,

f) detecting and/or quantitating the amount of nucleic acid in said sample by detecting and/or quantitating the amount of said at least one detectable species or the amount of said at least one binding species bound to said solid phase.

4. A method for the detection and/or quantitation of nucleic acid in a sample, which comprises:

a) mixing at least one labeled random primer at least 4 nucleotides in length having at least one binding species and optionally at least one detectable species, with a sample nucleic acid

b) adding at least one nucleic acid ligase

c) adding at least one NTP

d) incubating the mixture of step c), under conditions which allow said at least one nucleic acid ligase to be active,

e) contacting the mixture of step d) with at least one solid phase,

f) detecting and/or quantitating the amount of nucleic acid in said sample by detecting and/or quantitating the amount of said at least one detectable species or the amount of said at least one binding species bound to said solid phase.

13. A method as in

claim 4

or

5

wherein said at least one ligase is selected from then group consisting of Pfu DNA ligase, T4 DNA ligase, Taq DNA ligase, T4 RNA ligase, and E. coli DNA ligase.

5. A method for the detection and/or quantitation of nucleic acid in a sample, which comprises:

a) mixing at least one labeled random primer at least 4 nucleotides in length having at least one binding species and optionally at least one detectable species, with a sample nucleic acid

b) adding at least one nucleic acid ligase and at least one nucleic acid polymerase

c) adding at least one NTP

d) incubating the mixture of step c), under conditions which allow said at least one nucleic acid ligase and at least one nucleic acid polymerase to be active,

e) contacting the mixture of step d) with at least one solid phase,

f) detecting and/or quantitating the amount of nucleic acid in said sample by detecting and/or quantitating the amount of said at least one detectable species or the amount of said at least one binding species bound to said solid phase.

23. A method for the detection and/or quantitation of nucleic acid in a sample, which comprises:

a) mixing at least one random primer at least 4 nucleotides in length having at least one detectable species, with a sample nucleic acid,

b) adding at least one NTP having at least one binding species and optionally at least one NTP,

c) adding at least one nucleic acid polymerase,

d) incubating the mixture of step c), under conditions which allow said at least one nucleic acid polymerase to be active,

e) quantitating the amount of nucleic acid in said sample by detecting and/or quantitating the amount of said at least one detectable species or the amount of said at least one binding species.

28. A method as in

claim 23

,

24

, 25, 26 or 27 wherein said at least one detectable species is selected from the group consisting of biotin, avidin, streptavidin, antibody, antigen, lectin, receptor, ligand, hormone, nucleic acid sequence, mimitope, nucleic acid base pairing linear polymer, fluorescent molecule, electrochemiluminescent molecule, radioactive molecule, colored molecule, peroxidase, alkaline phosphatase and enzymes capable of producing a detectable species.

29. A method as in

claim 23

,

24

, 25 or 27 wherein said at least one nucleic acid polymerase is selected from the group consisting of Taq DNA polymerase, T4 DNA polymerase, Klenow fragment (3′

-5′

), Pfu DNA polymerase, Exo- Pfu DNA polymerase, E. coli DNA polymerase I, Klenow fragment of DNA polymerase I, MMLV reverse transcriptase and AMV reverse transcriptase.

30. A method as in

claim 23

,

24

, 25, 26 or 27 wherein said at least one NTP is selected from the group consisting of dATP, dGTP, dCTP, dUTP, dTTP, ATP, CTP, GTP, TTP, UTP inosineTP, propyne dCTP, propyne dUTP, 5-bromo dCTP, 5-iodo dUTP, 5-fluoro dUTP, O-6 methyl dGTP, 7-deaza dGTP, N-6 methyl-2′

-dATP, biotin-dATP, biotin-dCTP, biotin-dUTP, digoxigenin dUTP, digoxigenin UTP and biotin ddUTP.

31. A method as in

claim 23

,

24

, 25, 26 or 27 wherein said at least one random primer is 4-70 nucleotides.

32. A method as in

claim 23

,

24

, 25, 26 or 27 wherein said conditions comprise a solution with a pH between 5.5 and 9.5, a NTP concentration between 1 pM and 10 mM, a Mg2+ concentration between 0.05 mM and 500 mM, and a reducing agent concentration between 0 and 500 mM, where the sum of the molarities is between 1 M and 500 mM.

24. A method for the detection and/or quantitation of nucleic acid in a sample, which comprises:

a) mixing at least one random primer at least 4 nucleotides in length, having at least one binding species, with a sample nucleic acid,

b) adding at least one NTP having at least one detectable species and optionally at least one NTP,

c) adding at least one nucleic acid polymerase,

d) incubating the mixture of step c), under conditions which allow said at least one nucleic acid polymerase to be active,

e) quantitating the amount of nucleic acid in said sample by detecting and/or quantitating the amount of said at least one detectable species or the amount of said at least one binding species.

25. A method for the detection and/or quantitation of nucleic acid in a sample, which comprises:

a) mixing at least one random primer at least 4 nucleotides in length with a sample nucleic acid,

b) adding at least one NTP having at least one binding moiety and optionally at least one NTP having at least one label and optionally at least one NTP,

c) adding at least one nucleic acid polymerase,

d) incubating the mixture of step c), under conditions which allow said at least one nucleic acid polymerase to be active,

e) quantitating the amount of nucleic acid in said sample by detecting and/or quantitating the amount of said at least one label or the amount of said at least one binding moiety.

26. A method for the detection and/or quantitation of nucleic acid in a sample, which comprises:

a) mixing at least one labeled random primer at least 4 nucleotides in length having at least one binding species and optionally at least one detectable species, with a sample nucleic acid,

b) adding at least one nucleic acid ligase,

c) adding at least one NTP,

d) incubating the mixture of step b), under conditions which allow said at least one nucleic acid ligase to be active,

e) quantitating the amount of nucleic acid in said sample by detecting and/or quantitating the amount of said at least one detectable species or the amount of said at least one binding species.

33. A method as in

claim 26

or

27

wherein said at least one ligase is selected from then group consisting of Pfu DNA ligase, T4 DNA ligase, Taq DNA ligase, T4 RNA ligase, and E. coli DNA ligase.

27. A method for the detection and/or quantitation of nucleic acid in a sample, which comprises:

a) mixing at least one labeled random primer at least 4 nucleotides in length having at least one binding species and optionally at least one detectable species, with a sample nucleic acid,

b) adding at least one nucleic acid ligase and at least one nucleic acid polymerase,

c) adding at least one NTP,

d) incubating the mixture of step b), under conditions which allow said at least one nucleic acid ligase and at least one nucleic acid polymerase to be active,

e) quantitating the amount of nucleic acid in said sample by detecting and/or quantitating the amount of said at least one detectable species or the amount of said at least one binding species.

34. A kit comprising

a) a vial containing at least one random primer at least 4 nucleotides in length having at least one detectable species, and containing at least one NTP having at least one binding species and optionally at least one NTP,

b) a vial containing at least one nucleic acid polymerase, and

c) a vial containing at least one solid phase.

35. A kit as in

claim 34

where in component a) consists of a vial containing at least one random primer at least 4 nucleotides in length having at least one detectable species, and a vial containing at least one NTP having at least one binding species and optionally at least one NTP.

36. A kit comprising

a) a vial containing at least one random primer at least 4 nucleotides in length having at least one binding species, and containing at least one NTP having at least one detectable species and optionally at least one NTP,

b) a vial containing at least one nucleic acid polymerase, and

c) a vial containing at least one solid phase.

37. A kit as in

claim 36

where in component a) consists of a vial containing at least one random primer at least 4 nucleotides in length having at least one binding species, and a vial containing at least one NTP having at least one detectable species and optionally at least one NTP.