Assay methods for nucleic acid in a sample

• US 6,635,418 B2
• Filed: 02/13/1998
• Issued: 10/21/2003
• Est. Priority Date: 02/13/1998
• Status: Expired due to Fees

• Current Assignee: BioVeris Corporation (Roche Holding AG)
• Original Assignee: IGEN International, Inc. (Roche Holding AG)
• Inventors: Corcoran, Marta L., Rao, Savitha M., Heroux, Jeffrey A.
• Primary Examiner(s): Siew, Jeffrey
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US09/023,483
• Publication Number:
  US 20010014446A1
• Time in Patent Office: 2,076 Days
• Field of Search: 435/91.2, 435/91.52, 435/91.51, 435/403, 536/25.32, 536/26.6
• US Class Current: 435/6.1
• 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 determining total 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 which may contain nucleic acid,

b) adding at least one nucleotide triphosphate having at least one binding species and optionally at least one second nucleotide triphosphate,

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) measuring total nucleic acid in said sample by measuring the total amount of said at least one detectable species bound to said solid phase, and

g) determining whether the total nucleic acid in said sample is higher or lower than a threshold amount of contamination, wherein the threshold amount of contamination is equal to or less than 100 pg.

6. A method as in claim 1, 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, 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, wherein said at least one nucleic acid polymerase is selected from the group consisting of Taq DNA polymerase, T4 DNA polymerase, Klenow fragment, 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, 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, wherein said at least one nucleotide triphosphate 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, wherein said at least one random primer is 4-70 nucleotides.

12. A method as in claim 1, wherein said conditions comprise a solution with a pH between 5.5 and 9.5, a nucleotide triphosphate 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, wherein 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 detectable species 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′

) of 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 nucleotide triphosphate 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.

29. A method as in claim 1, wherein said at least one nucleic acid polymerase is selected from the group consisting of Taq DNA polymerase, T4 DNA polymerase, Klenow fragment, 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.

37. The method of claim 1, wherein said method is capable of detecting total nucleic acid in amounts as low as 5 pg.

50. The method of claim 1, wherein said method is capable of detecting DNA fragments shorter than 800 basepairs.

2. A method for determining total 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 which may contain nucleic acid

b) adding at least one nucleotide triphosphate having at least one detectable species and optionally at least one second nucleotide triphosphate,

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) measuring total nucleic acid in said sample by measuring the total amount of said at least one detectable species bound to said solid phase, and

g) determining whether the total nucleic acid in said sample is higher or lower than a threshold amount of contamination, wherein the threshold amount of contamination is equal to or less than 100 pg.

38. The method of claim 2, wherein said method is capable of detecting total nucleic acid in amounts as low as 5 pg.

51. The method of claim 2, wherein said method is capable of detecting total DNA fragments shorter than 800 basepairs.

3. A method for determining total nucleic acid in a sample, which comprises:

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

b) adding at least one nucleotide triphosphate having at least one binding species and optionally at least one nucleotide triphosphate having at least one detectable species and optionally at least one second nucleotide triphosphate,

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) measuring total nucleic acid in said sample by measuring the total amount of said at least one detectable species or the amount of said at least one binding species bound to said solid phase, and

g) determining whether the total nucleic acid in said sample is higher or lower than a threshold amount of contamination, wherein the threshold amount of contamination is equal to or less than 100 pg.

39. The method of claim 3, wherein said method is capable of detecting total nucleic acid in amounts as low as 5 pg.

52. The method of claim 3, wherein said method is capable of detecting total DNA fragments shorter than 800 basepairs.

4. A method for determining total nucleic acid in a sample, which comprises:

a) mixing at least one first labeled random primer at least 4 nucleotides in length having at least one binding species and at least one second random primer at least 4 nucleotides in length having at least one detectable species, with a sample which may contain nucleic acid,

b) adding at least one nucleic acid ligase,

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

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

e) measuring total nucleic acid in said sample by measuring the total amount of said at least one detectable species or the amount of said at least one binding species bound to said solid phase, and

f) determining whether the total nucleic acid in said sample is higher or lower than a threshold amount of contamination, wherein the threshold amount of contamination is equal to or less than 100 pg.

13. A method as in claim 4, wherein said at least one ligase is selected from the group consisting of Pfu DNA ligase, T4 DNA ligase, Taq DNA ligase, T4 RNA ligase, and E. coli DNA ligase.

40. The method of claim 4, wherein said method is capable of detecting total nucleic acid in amounts as low as 5 pg.

53. The method of claim 4, wherein said method is capable of detecting total DNA fragments shorter than 800 basepairs.

5. A method for determining total nucleic acid in a sample, which comprises:

a) mixing at least one first labeled random primer at least 4 nucleotides in length having at least one binding species and at least one second random primer at least 4 nucleotides in length having at least one detectable species, with a sample which may contain nucleic acid,

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

c) 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,

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

c) measuring total nucleic acid in said sample by measuring the total amount of said at least one detectable species or the amount of said at least one binding bound to said solid phase, and

f) determining whether the total nucleic acid in said sample is higher or lower than a threshold amount of contamination, wherein the threshold amount of contamination is equal to or less than 100 pg.

41. The method of claim 5, wherein said method is capable of detecting total nucleic acid in amounts as low as 5 pg.

54. The method of claim 5, wherein said method is capable of detecting total DNA fragments shorter than 800 basepairs.

23. A method for determining total 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 which may contain nucleic acid,

b) adding at least one nucleotide triphosphate having at least one binding species and optionally at least one second nucleotide triphosphate,

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) measuring total nucleic acid in said sample by measuring the total amount of said at least one detectable species or the amount of said at least one binding species, and

f) determining whether the total nucleic acid in said sample is higher or lower than a threshold amount of contamination, wherein the threshold amount of contamination is equal to or less than 100 pg.

28. A method as in claim 23, 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.

30. A method as in claim 23, wherein said at least one nucleotide triphosphate 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, wherein said at least one random primer is 4-70 nucleotides.

32. A method as in claim 23, wherein said conditions comprise a solution with a pH between 5.5 and 9.5, a nucleotide triphosphate 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.

42. The method of claim 23, wherein said method is capable of detecting total nucleic acid in amounts as low as 5 pg.

55. The method of claim 23, wherein said method is capable of detecting DNA fragments shorter than 800 basepairs.

24. A method for determining total 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 which may contain nucleic acid,

b) adding at least one nucleotide triphosphate having at least one detectable species and optionally at least one second nucleotide triphosphate,

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) measuring total nucleic acid in said sample by measuring the total amount of said at least one detectable species or the amount of said at least one binding species, and

f) determining whether the total nucleic acid in said sample is higher or lower than a threshold amount of contamination, wherein the threshold amount of contamination is equal to or less than 100 pg.

43. The method of claim 24, wherein said method is capable of detecting total nucleic acid in amounts as low as 5 pg.

56. The method of claim 24, wherein said method is capable of detecting DNA fragments shorter than 800 basepairs.

25. A method for determining total nucleic acid in a sample, which comprises:

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

b) adding at least one nucleotide triphosphate having at least one binding moiety and optionally at least one second nucleotide triphosphate having at least one label and optionally at least one nucleotide triphosphate,

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) measuring total nucleic acid in said sample by measuring the total amount of said at least one label or the amount of said at least one binding moiety, and

f) determining whether the total nucleic acid in said sample is higher or lower than a threshold amount of contamination, wherein the threshold amount of contamination is equal to or less than 100 pg.

44. The method of claim 25, wherein said method is capable of detecting total nucleic acid in amounts as low as 5 pg.

57. The method of claim 25, wherein said method is capable of detecting DNA fragments shorter than 800 basepairs.

26. A method for determining total 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 second random primer at least 4 nucleotides in length having at least one detectable species, with a sample nucleic acid,

b) adding at least one nucleic acid ligase,

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

d) measuring total nucleic acid in said sample by measuring the total amount of said at least one detectable species or the amount of said at least one binding species, and

e) determining whether the total nucleic acid in said sample is higher or lower than a threshold amount of contamination, wherein the threshold amount of contamination is equal to or less than 100 pg.

33. A method as in claim 26, wherein said at least one ligase is selected from the group consisting of Pfu DNA ligase, T4 DNA ligase, Taq DNA ligase, T4 RNA ligase, and E. coli DNA ligase.

45. The method of claim 26, wherein said method is capable of detecting total nucleic acid in amounts as low as 5 pg.

58. The method of claim 26, wherein said method is capable of detecting DNA fragments shorter than 800 basepairs.

27. A method for determining total 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 second random primer at least 4 nucleotides in length having at least one detectable species, with a sample which may contain nucleic acid,

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

c) adding at least one nucleotide triphosphate,

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) measuring total nucleic acid in said sample by measuring the total amount of said at least one detectable species or the amount of said at least one binding species, and

f) determining whether the total nucleic acid in said sample is higher or lower than a threshold amount of contamination, wherein the threshold amount of contamination is equal to or less than 100 pg.

46. The method of claim 27, wherein said method is capable of detecting total nucleic acid in amounts as low as 5 pg.

59. The method of claim 27, wherein said method is capable of detecting DNA fragments shorter than 800 basepairs.

34. A method for determining total nucleic acid in a sample, which comprises:

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

b) adding at least one nucleotide triphosphate having at least one second label and optionally at least one second nucleotide triphosphate,

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) measuring total nucleic acid in said sample by measuring the total amount of said at least one first label or the amount of said at least one second label, and

f) determining whether the total nucleic acid in said sample is higher or lower than a threshold amount of contamination, wherein the threshold amount of contamination is equal to or less than 100 pg.

47. The method of claim 34, wherein said method is capable of detecting total nucleic acid in amounts as low as 5 pg.

60. The method of claim 34, wherein said method is capable of detecting DNA fragments shorter than 800 basepairs.

35. A method for determining total 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 first label species and optionally at least one second random primer at least 4 nucleotides in length having at least one second label, with a sample which may contain nucleic acid,

b) adding at least one nucleic acid ligase,

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

d) measuring total nucleic acid in said sample by measuring the total amount of said at least one first label or the amount of said at least one second label, and

e) determining whether the total nucleic acid in said sample is higher or lower than a threshold amount of contamination, wherein the threshold amount of contamination is equal to or less than 100 pg.

48. The method of claim 35, wherein said method is capable of detecting total nucleic acid in amounts as low as 5 pg.

61. The method of claim 35, wherein said method is capable of detecting DNA fragments shorter than 800 basepairs.

36. A method for determining total 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 first label and optionally at least one second random primer at least 4 nucleotides in length having at least one second label, with a sample which may contain nucleic acid

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

c) adding at least one nucleotide triphosphate,

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) measuring total nucleic acid in said sample by measuring the total amount of said at least one first label or the amount of said at least one second label, and

f) determining whether the total nucleic acid in said sample is higher or lower than a threshold amount of contamination, wherein the threshold amount of contamination is equal to or less than 100 pg.

49. The method of claim 36, wherein said method is capable of detecting total nucleic acid in amounts as low 5 pg.

62. The method of claim 36, wherein said method is capable of detecting DNA fragments shorter than 800 basepairs.