NUCLEOTIDE TRANSIENT BINDING FOR SEQUENCING METHODS

• US 20100330570A1
• Filed: 05/28/2010
• Published: 12/30/2010
• Est. Priority Date: 06/05/2009
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: VANDER HORN, Peter B., Temirov, Jamshid, Nishimura, Stefanie Yukiko, Wang, Yufang, Nikiforov, Theo, Previte, Michael, Rosenblum, Barnett B., Sun, Hongye, Peris, Marian, Wang, Hongyi, Luo, Guobin, Phelan, Michael, Zhou, Zhaohui, Chen, Cheng-Yao
• Granted Patent: US 8,632,975 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/137 Metal/ion, e.g. metal label
• CPC Class Codes: C12Q 2565/30 Detection characterised by liberation or release of label
• CPC Class Codes: C12Q 2565/632 being a surface enhanced, e.g. resonance, Raman spectrometer
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: G01N 2500/00 Screening for compounds of potential therapeutic value

Abstract

Provided herein are compositions and systems for use in polymerase-dependent, nucleotide transient-binding methods. The methods are useful for deducing the sequence of a template nucleic acid molecule and single nucleotide polymorphism (SNP) analyses. The methods rely on the fact that the polymerase transient-binding time for a complementary nucleotide is longer compared to that of a non-complementary nucleotide. The labeled nucleotides transiently-binds the polymerase in a template-dependent manner, but does not incorporate. The methods are conducted under any reaction condition that permits transient binding of a complementary or non-complementary nucleotide to a polymerase, and inhibits nucleotide incorporation.

Claims

1. A method for identifying a nucleotide bound to a polymerase, comprising:

a) contacting at least one type of a labeled nucleotide to an immobilized complex having a first polymerase bound to a template nucleic acid molecule that is bound to a polymerization initiation site, under suitable conditions to transiently-bind the at least one type of labeled nucleotide to the polymerase in a nucleic acid template-dependent manner and to inhibit nucleotide polymerization by the polymerase;

b) detecting the transiently-bound labeled nucleotide; and

c) identifying the labeled nucleotide transiently-bound to the polymerase.

2. The method of claim 1, wherein the suitable conditions in step (a) comprise:

(i) reducing the levels or omission of a metal cation that permits nucleotide incorporation and/or addition of a cation that inhibits nucleotide incorporation;

(ii) the polymerase selectively binds the nucleotide in a template-dependent manner and exhibits reduced nucleotide incorporation activity;

(iii) the at least one type of labeled nucleotide is a labeled non-incorporatable nucleotide; and

/or (iv) the polymerization initiation site is a non-extendible polymerization initiation site.

3. The method of claim 1, wherein the suitable conditions in step (a) comprise:

(i) metal cations present at a concentration that inhibits nucleotide incorporation;

(ii) the polymerase selectively binds the nucleotide in a template-dependent manner and exhibits reduced nucleotide incorporation activity;

(iii) the at least one type of labeled nucleotide is a labeled non-incorporatable nucleotide; and

/or (iv) the polymerization initiation site is a non-extendible polymerization initiation site.

4. The method of claim 1 further comprising:

a) removing the transiently-bound nucleotide; and

b) contacting the complex with at least one type of a second nucleotide under suitable conditions for the polymerase to polymerize the nucleotide.

5. The method of claim 4, wherein the suitable conditions in step (b) comprise:

(i) including a metal cation that permits nucleotide incorporation and/or reducing the levels or omission of a cation that inhibits nucleotide incorporation;

(ii) the polymerase selectively binds the nucleotide in a template-dependent manner and polymerizes the bound nucleotide;

(iii) the at least one type of a second nucleotide is an incorporatable nucleotide; and

/or (iv) the polymerization initiation is an extendible polymerization initiation site.

6. The method of claim 1, wherein the polymerization initiation site is a 3′

terminal end of a nucleic acid primer molecule or a 3′

terminal end of a self-priming template nucleic acid molecule.

7. The method of claim 6 further comprising the steps of:

a) removing the first polymerase and the transiently-bound nucleotide so that the template nucleic acid molecule, the nucleic acid primer molecule or the self-priming template nucleic acid molecule remains immobilized to the surface;

b) binding the remaining template nucleic acid molecule, the nucleic acid primer molecule or the self-priming template nucleic acid molecule, with a second polymerase; and

c) contacting the second polymerase with at least one type of nucleotide under suitable conditions for the second polymerase to polymerize the nucleotide.

8. The method of claim 6, wherein the suitable conditions in step (c) comprise:

(i) including a metal cation that permits nucleotide incorporation and/or reducing the levels or omission of a cation that inhibits nucleotide incorporation;

(ii) the polymerase selectively binds the nucleotide in a template-dependent manner and polymerizes the bound nucleotide;

(iii) the at least one type of second nucleotide is an incorporatable nucleotide; and

/or (iv) the polymerization initiation site is an extendible polymerization initiation site.

9. The method of claim 1, wherein the polymerization initiation site is base-paired to the template nucleic acid molecule.

10. The method of claim 1, wherein the polymerization initiation site comprises an extendible terminal 3′

OH group or a non-extendible terminal group.

11. The method of claim 1, wherein the polymerization initiation site comprises a terminal 3′

OH group of the nucleic acid primer molecule or a terminal 3′

OH group of a self-priming template nucleic acid molecule.

12. The method of claim 11, wherein the template nucleic acid molecule, nucleic acid primer molecule, or self-priming template nucleic acid molecule is immobilized to a surface

13. The method of claim 1, wherein the labeled nucleotide is excited with electromagnetic radiation.

14. The method of claim 2, wherein the cation that inhibits nucleotide incorporation is calcium, scandium, titanium, vanadium, chromium, iron, cobalt, nickel, copper, zinc, gallium, germanium, arsenic, selenium, rhodium, or strontium.

15. The method of claim 2, wherein the cation that permits nucleotide incorporation is magnesium or manganese.

16. The method of claim 1, wherein the first polymerase binds the labeled nucleotide in a nucleic acid template-dependent manner and exhibits reduced nucleotide polymerization activity.

17. The method of claim 1, wherein the first polymerase is a DNA-dependent polymerase, RNA-dependent polymerase, or a reverse transcriptase.

18. The method of claim 1, wherein the first polymerase is an RB69 polymerase, phi29 polymerase, B103 polymerase, or Klenow fragment polymerase.

19. The method of claim 1, wherein the first polymerase is an RB69 polymerase according to SEQ ID NO:

5.

20. The method of claim 4, wherein the second polymerase is the same type or a different type as the first polymerase.

21. The method of claim 1, wherein the one type of labeled nucleotide in step (a) comprises 3-10 phosphate groups.

22. The method of claim 1, wherein the one type of labeled nucleotide in step (a) comprise an adenosine, guanosine, cytidine, thymidine, or uridine nucleotide.

23. The method of claim 1, wherein the labeled nucleotide comprises the label linked to the base of the nucleotide or linked to the terminal phosphate group of the nucleotide.

24. The method of claim 23, wherein the label comprises a fluorescent dye.

25. The method of claim 23, wherein the label comprises an energy transfer acceptor reporter moiety.

26. The method of claim 22, wherein the adenosine, guanosine, cytidine, thymidine, or uridine nucleotide comprise different types of labels.

27. The method of claim 1, wherein the complex is contacted with at least two types of labeled nucleotides in step (a).

28. The method of claim 27, wherein the at least two types of nucleotides comprise different types of labels.

29. The method of claim 1, wherein the polymerase comprises an energy transfer donor reporter moiety.

30. The method of claim 29, wherein the energy transfer donor reporter moiety is an inorganic nanoparticle or a fluorophore.

31. The method of claim 1, wherein the polymerase comprises an energy transfer donor reporter moiety and the transiently-bound nucleotide comprises an energy transfer acceptor reporter moiety.

32. The method of claim 31, wherein the transiently-bound labeled nucleotide produces a FRET signal.

33. The method of claim 1, wherein the signal from the transiently-bound labeled nucleotide is optically detectable.

34. The method of claim 1, wherein the one type of labeled nucleotide in step (a) comprises a labeled non-incorporatable nucleotide.

35. The method of claim 34, wherein the labeled non-incorporatable nucleotide is an adenosine, guanosine, cytidine, thymidine, or uridine nucleotide.

36. The method of claim 34, wherein the label comprises a fluorescent dye.

37. The method of claim 34, wherein the label comprises an energy transfer acceptor reporter moiety.

38. The method of claim 34, wherein the adenosine, guanosine, cytidine, thymidine, or uridine nucleotide comprise different labels.

39. The method of claim 34, wherein the polymerase comprises an energy transfer donor reporter moiety.

40. The method of claim 34, wherein the energy transfer donor reporter moiety is an inorganic nanoparticle or a fluorophore.

41. The method of claim 34, wherein the polymerase comprises an energy transfer donor reporter moiety and the non-incorporatable nucleotide comprises an energy transfer acceptor reporter moiety.

42. The method of claim 34, wherein the signal emitted by the transiently-bound labeled nucleotide is a FRET signal.

43. The method of claim 10, wherein the non-extendible terminal group comprises an inhibitor moiety which inhibits incorporation of a subsequent in-coming nucleotide.

44. The method of claim 43, wherein the inhibitor moiety is removable via an enzymatic, heat, chemical, or light cleavage reaction.

45. The method of claim 1, wherein the template nucleic acid molecule is a DNA, RNA, or DNA/RNA hybrid molecule.

46. A method for identifying a nucleotide bound to a polymerase, comprising:

a) contacting at least one type of a labeled nucleotide to an immobilized complex having a polymerase bound to a template nucleic acid molecule which is bound to a polymerization initiation site, under suitable conditions to transiently-bind the at least one type of labeled nucleotide to the polymerase in a nucleic acid template-dependent manner and to inhibit nucleotide polymerization by the polymerase;

b) exciting the first labeled nucleotide with an excitation source;

c) detecting a signal, or a change in a signal, from the transiently-bound labeled nucleotide; and

d) identifying the nucleotide transiently-bound to the polymerase.

e) removing the transiently-bound nucleotide;

f) contacting the complex with at least one type of nucleotide under suitable conditions for the polymerase to polymerize the nucleotide; and

g) repeating steps (a)-(e).

47. The method of claim 46, wherein the suitable conditions in step (a) comprise:

(i) reducing the levels or omission of a metal cation that permits nucleotide incorporation and/or addition of a cation that inhibits nucleotide incorporation;

(ii) using a polymerase which selectively binds the nucleotide in a template-dependent manner and exhibits reduced nucleotide incorporation activity;

(iii) using at least one type of labeled nucleotide which is a labeled non-incorporatable nucleotide; and

/or (iv) using a polymerization initiation site which is a non-extendible polymerization initiation site.

48. The method of claim 46, wherein the suitable conditions in step (e) comprise:

(i) including a metal cation that permits nucleotide incorporation and/or reducing the levels or omission of a cation that inhibits nucleotide incorporation;

(ii) using a polymerase which selectively binds the nucleotide in a template-dependent manner and polymerizes the bound nucleotide;

(iii) using at least one type of incorporatable nucleotide; and

/or (iv) using a polymerization initiation site having an extendible polymerization initiation site.

49. A method for identifying a nucleotide bound to a polymerase, comprising:

a) contacting at least one type of a labeled nucleotide to an immobilized complex having a polymerase bound to a template nucleic acid molecule which is base-paired to a nucleic acid primer molecule or the immobilized complex having a polymerase bound to a self-priming template nucleic acid molecule, under suitable conditions to transiently-bind the at least one type of labeled nucleotide to the polymerase in a nucleic acid template-dependent manner and to inhibit nucleotide polymerization by the polymerase;

b) exciting the first labeled nucleotide with an excitation source;

c) detecting a signal, or a change in a signal, from the transiently-bound labeled nucleotide; and

d) identifying the nucleotide transiently-bound to the polymerase.

e) removing the polymerase and the transiently-bound nucleotide so that the template nucleic acid molecule, primer nucleic acid molecule or self-priming template nucleic acid molecule remains immobilized to the surface;

f) binding the remaining template nucleic acid molecule with a second polymerase;

g) contacting the second polymerase with at least one type of nucleotide under suitable conditions for the second polymerase to polymerize the nucleotide; and

h) repeating steps (a)-(f).

50. The method of claim 49, wherein the suitable conditions in step (a) comprise:

(i) reducing the levels or omission of a metal cation that permits nucleotide incorporation and/or addition of a cation that inhibits nucleotide incorporation;

(ii) using a polymerase which selectively binds the nucleotide in a template-dependent manner and exhibits reduced nucleotide incorporation activity;

(iii) using at least one type of labeled nucleotide which is a labeled non-incorporatable nucleotide; and

/or (iv) using a polymerization initiation site which is a non-extendible polymerization initiation site.

51. The method of claim 49, wherein the suitable conditions in step (f) comprise:

(i) including a metal cation that permits nucleotide incorporation and/or reducing the levels or omission of a cation that inhibits nucleotide incorporation;

(ii) using a polymerase which selectively binds the nucleotide in a template-dependent manner and polymerizes the bound nucleotide;

(iii) using at least one type of incorporatable nucleotide; and

/or (iv) using a polymerization initiation site having an extendible polymerization initiation site.

52. A method for identifying a nucleotide bound to a polymerase, comprising:

a) contacting a labeled nucleotide to an immobilized complex having a first polymerase bound to a template nucleic acid molecule that is bound to a polymerization initiation site, wherein the labeled nucleotide is transiently-bound to the complex;

b) detecting the transiently-bound labeled nucleotide; and

c) identifying the labeled nucleotide transiently-bound to the polymerase.

53. The method of claim 52, wherein the contacting in step a) comprises:

(i) a metal cation present at a concentration that inhibits nucleotide incorporation;

(ii) the polymerase selectively binds the labeled nucleotide in a template-dependent manner and exhibits reduced nucleotide incorporation activity;

(iii) the labeled nucleotide is a non-incorporatable nucleotide; and

/or (iv) the polymerization initiation site is a non-extendible polymerization initiation site.

54. The method of claim 53, wherein the metal cation that inhibits nucleotide incorporation is calcium.

55. The method of claim 52 further comprising:

a) removing the transiently-bound nucleotide; and

b) contacting the complex with a nucleotide under suitable conditions for the polymerase to polymerize the nucleotide.

56. The method of claim 54, wherein the contacting in step a) comprises:

(i) a metal cation present at a concentration that permits nucleotide incorporation;

(ii) the polymerase selectively binds the nucleotide in a template-dependent manner and exhibits nucleotide incorporation activity;

(iii) the nucleotide is an incorporatable nucleotide; and

/or (iv) the polymerization initiation site is an extendible polymerization initiation site.

57. The method of claim 56, wherein the metal cation that permits nucleotide incorporation is magnesium or manganese.

58. A system comprising:

a polymerase which is complexed with a template nucleic acid molecule which is bound to a polymerization initiation site, and a nucleotide which is transiently bound to the active site of the polymerase in a template-dependent manner.

59. The system of claim 58, wherein the transiently-bound nucleotide is inhibited from incorporation onto the polymerization initiation site by suitable conditions which include:

(i) reducing the levels or omission of a metal cation that permits nucleotide incorporation and/or addition of a cation that inhibits nucleotide incorporation;

(ii) using a polymerase which selectively binds the nucleotide in a template-dependent manner and exhibits reduced nucleotide incorporation activity;

(iii) using at least one type of labeled nucleotide which is a labeled non-incorporatable nucleotide; and

/or (iv) using a polymerization initiation site which is a non-extendible polymerization initiation site.

60. The system of claim 58, wherein the polymerase comprises the amino acid sequence according to SEQ ID NO:

5.