METHODS FOR SYNCHRONIZING NUCLEIC ACID MOLECULES

• US 20170044601A1
• Filed: 08/12/2016
• Published: 02/16/2017
• Est. Priority Date: 08/13/2015
• Status: Active Grant

• Current Assignee: Centrillion Technology Holdings Corporation (Centrillion Technology, Inc.)
• Original Assignee: Centrillion Technology Holdings Corporation (Centrillion Technology, Inc.)
• Inventors: Crnogorac, Filip, Zhou, Wei
• Granted Patent: US 10,584,378 B2
• Time in Patent Office: Days
• 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 2535/122 Massive parallel sequencing

Abstract

The methods, systems, kits and reagents provided herein relate to the synchronization of dephased/prephased nucleic acid molecules during sequencing reactions. The methods, systems, kits and reagents provided herein can be utilized to improve the efficiency and accuracy of sequencing technologies.

Claims

1-19. -19. (canceled)

20. A method of improving a length of accurate base calls in a sequencing read of a sequencing reaction, said method comprising performing one or more chemical synchronization steps on a plurality of sequencing products, thereby increasing said length of accurate base calls in said sequencing read of said sequencing reaction.

21. The method of claim 20, wherein said length of accurate base calls in said sequencing read of said sequencing reaction is increased by at least 10 base pairs as compared to a length of accurate base calls in a sequencing read of a sequencing reaction performed in the absence of one or more synchronization steps.

24. The method of claim 20, wherein each of said one or more synchronization steps comprises extending said plurality of sequencing products in the presence of a set of up to three different nucleotides selected from the group consisting of:

dATP, dCTP, dGTP, dTTP and dUTP.

29. The method of claim 20, further comprising, prior to performing said one or more synchronization steps, performing one or more successive rounds of sequencing, wherein each round of sequencing comprises (i) extending a plurality of sequencing primers using a plurality of target nucleic acids as template in the presence of one or more labeled nucleotides to generate said plurality of sequencing products;

and (ii) determining a nucleic acid sequence of said plurality of sequencing products.

31. The method of claim 29, further comprising, after performing said one of more synchronization steps, repeating, one or more times, one or more rounds of sequencing followed by one or more synchronization steps.

33. The method of claim 20, wherein said method of improving said length of accurate base calls comprises performing at least four synchronization steps.

35. The method of claim 29, wherein said performing said one or more successive rounds of sequencing comprises performing from 100 to 200 successive rounds of sequencing.

64. The method of claim 20, wherein the increased length of accurate base calls comprises a series of noncontiguous accurate base calls.

22-23. -23. (canceled)

25-28. -28. (canceled)

30. (canceled)

32. (canceled)

34. (canceled)

36-38. -38. (canceled)

39. A method of synchronizing one or more unsynchronized sequencing products, said method comprising:

(a) performing one or more synchronization steps on a plurality of sequencing products, said plurality of sequencing products comprising said one or more unsynchronized sequencing products, wherein each of said one or more synchronization steps comprises;

(i) contacting said plurality of sequencing products with a first set of up to three different nucleotides selected from the group consisting of;

dATP, dTTP, dCTP, dGTP and dUTP;

(ii) extending said plurality of sequencing products with a DNA polymerase; and

(iii) optionally, removing said first set of up to three different nucleotides,

thereby synchronizing said one or more unsynchronized sequencing products.

40. The method of claim 39, wherein a consecutive synchronization step of said one or more synchronization steps comprises:

contacting said plurality of sequencing products with a second set of up to three different nucleotides selected from the group consisting of;

dATP, dTTP, dCTP, dGTP and dUTP, wherein said second set of nucleotides is different from said first set of nucleotides.

44. The method of claim 39, wherein said set of up to three different nucleotides further comprises a reversible terminator nucleotide.

45. The method of claim 44, wherein after each synchronization step of said one or more synchronization steps, said reversible terminator nucleotide is deblocked and said plurality of sequencing products are made ready for further extension.

49. The method of claim 39, wherein, after performing said one or more synchronization steps, at least 95% of said sequencing products are synchronized.

50. The method of claim 39, wherein, after performing said one or more synchronization steps, said sequencing products having a chastity score of at least 0.85.

52. The method of claim 39, further comprising, removing said set of up to three different nucleotides after each synchronization step (i) by washing or (ii) by the use of a nucleotide degrading enzyme.

63. The method of claim 39, wherein said one or more synchronization steps comprises eight consecutive synchronization steps, said eight consecutive synchronization steps comprising:

contacting said plurality of sequencing products with dCTP, dGTP and dTTP;

contacting said plurality of sequencing products with dATP, dTTP, and dGTP;

contacting said plurality of sequencing products with dATP, dCTP, and dTTP;

contacting said plurality of sequencing products with dATP, dCTP and dGTP;

contacting said plurality of sequencing products with dATP, dCTP, and dTTP;

contacting said plurality of sequencing products with dATP, dTTP, and dGTP;

contacting said plurality of sequencing products with dCTP, dGTP and dTTP; and

contacting said plurality of sequencing products with dATP, dCTP and dGTP.

41-43. -43. (canceled)

46-48. -48. (canceled)

51. (canceled)

53-54. -54. (canceled)

55. A kit for sequencing a target nucleic acid molecule, said kit comprising:

(a) a primer hybridizable to said target nucleic acid molecule,

(b) one or more labeled nucleotides; and

(c) one or more sets of up to three different nucleotides selected from the group consisting of;

dATP, dTTP, dCTP, dGTP and dUTP.

57. The kit of claim 55, further comprising a pyrophosphatase.

58. The kit of claim 55, further comprising an apyrase.

61. The kit of claim 55, wherein said one or more sets of up to three different nucleotides further comprises a reversible terminator nucleotide.

56. (canceled)

59-60. -60. (canceled)

62. (canceled)