METHODS AND COMPOSITIONS FOR DETECTING GENETIC MATERIAL

• US 20110159499A1
• Filed: 11/25/2010
• Published: 06/30/2011
• Est. Priority Date: 11/25/2009
• Status: Abandoned Application

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: QuantaLife, Inc. (Bio-Rad Laboratories Incorporated)
• Inventors: Ness, Kevin, HINDSON, Benajamin, Saxonov, Serge, Colston, Billy, Belgrader, Phillip, Lucero, Michael
• Application Number:
  US12/954,634
• Publication Number:
  US 20110159499A1
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2563/161 Vesicles, e.g. liposome

Abstract

This invention provides compositions and methods for detecting differences in copy number of a target polynucleotide. In some cases, the methods and compositions provided herein are useful for diagnosis of fetal genetic abnormalities, when the starting sample is maternal tissue (e.g., blood, plasma). The methods and materials described apply techniques for allowing detection of small, but statistically significant, differences in polynucleotide copy number.

Claims

1. A method of detecting copy number of a target polynucleotide within a population of genetic material comprising:

a. binding a first ligation probe to a first target polynucleotide;

b. binding a second ligation probe to a second target polynucleotide;

c. subjecting said first and second ligation probes to a ligation reaction to obtain one or more ligated products;

d. partitioning said one or more ligated products into two or more partitions;

e. amplifying a region within said one or more ligated products to obtain amplified products;

f. determining a number of said partitions that contain said amplified products; and

g. calculating a copy number of said first target polynucleotide based on said number of said partitions.

2. The method of claim 1, wherein said target polynucleotide is not partitioned into said two or more partitions.

3. The method of claim 1, wherein during the amplification process said two or more partitions remain substantially intact.

4. The method of claim 1, wherein during said determining of step (f), said two or more partitions remain substantially intact.

5. The method of claim 1, wherein said first and second ligation probes are each designed to bind to said first target polynucleotide

7. The method of claim 1, wherein said partitioning of step (d) does not comprise partitioning said target polynucleotide molecule.

8. The method of claim 1, wherein said two or more partitions comprise an amplification reaction that is initiated from said ligated product.

9. The method of claim 1, wherein said first ligation probe is designed to bind to a polynucleotide sequence that is conserved between individuals within a species.

10. The method of claim 1, wherein said partitions are aqueous droplets present within a mixture of at least two immiscible fluids.

11. The method of claim 10, wherein a continuous oil phase comprises said aqueous droplets.

12. The method of claim 1, further comprising binding at least four ligation probes to said first target polynucleotide.

13. The method of claim 1, further comprising binding at least four ligation probes to said first target polynucleotide and at least four ligation probes to said second target polynucleotide.

14. The method of claim 1, wherein said first ligation probe is designed to bind to a first region within said first target polynucleotide and said second ligation probe is designed to bind to a second region within said first target polynucleotide, wherein said first and second regions do not have identical sequences.

15. The method of claim 1, wherein said first target polynucleotide is not identical to said second target polynucleotide

16. The method of claim 15, wherein said first target polynucleotide is a test chromosome and said second target polynucleotide is a reference chromosome.

17. The method of claim 16, wherein said test chromosome is selected from the group consisting of:

chromosome 21, chromosome 13, chromosome 18, and the X chromosome.

18. The method of claim 1, wherein said first target polynucleotide is a chromosome selected from the group consisting of chromosome 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, X, and Y.

19. The method of claim 1, wherein said first target polynucleotide is a segment of a chromosome.

20. The method of claim 19, wherein said segment of a chromosome is associated with fetal aneuploidy.

21. The method of claim 1, wherein said ligation reaction results in the ligation of a 5′

region of said first ligation probe to a 3′

region of said first ligation probe to obtain a circular ligated product.

22. The method of claim 1, wherein said ligation reaction results in the ligation of the 5′

region of said first ligation probe to the 3′

region of said second ligation probe to obtain a linear ligated product comprising at least a portion of said first and second ligation probes.

23. The method of claim 21, wherein said 5′

region and said 3′

region of said first ligation probe are each designed to bind adjacent sequences within said first target polynucleotide.

24. The method of claim 21, wherein said 5′

region and said 3′

region of said first ligation probe are each designed to bind neighboring sequences within said first target polynucleotide

25. The method of claim 24, wherein said neighboring sequences are separated by at least one nucleotide.

26. The method of claim 25, wherein said ligation reaction further comprises a template-driven gap fill reaction to incorporate nucleotides in the region between said 5′

region and said 3′

region of said first ligation probe.

27. The method of claim 1, wherein said first ligation probe comprises a site cleavable by an enzyme.

28. The method of claim 27, wherein said site cleavable by an enzyme comprises one or more uracils.

30. The method of claim 1, wherein said first ligation probe is a padlock probe.

31. The method of claim 1, wherein said first ligation probe is a molecular inversion probe.

32. The method of claim 1, further comprising performing an enzymatic reaction to remove linear polynucleotides.

33. The method of claim 1, further comprising performing an enzymatic reaction to remove single-stranded polynucleotides.

34. The method of claim 1, wherein said first ligation probe is conjugated to a first signaling agent and wherein said second ligation probe is conjugated a second signaling agent.

35. The method of claim 34, wherein said first signaling agent is a fluorescent marker of a first color and said second signaling agent is a fluorescent marker of a second color.

36. The method of claim 35, wherein said determining of step (f) comprises detecting said first ligation probe with a first signaling agent and detecting said second ligation probe with a second signaling agent.

37. The method of claim 36, wherein said first ligation probe comprises a plurality of ligation probes, wherein each probe is directed to a different region of a first chromosome, and wherein said second ligation probe comprises a plurality of ligation probes, wherein each probe is directed to a different region of a second chromosome.

38. The method of claim 35, wherein said first target polynucleotide is a test chromosome and said second target polynucleotide is a reference chromosome.

39. The method of claim 14, wherein said first and second ligation probes are conjugated to a signaling agent with the same signaling color.

29. The method of 27, wherein said site cleavable by an enzyme comprises a restriction site.

40. A method of detecting copy number of a target polynucleotide within a population of genetic material comprising:

a. binding a first ligation probe to a first target polynucleotide;

b. binding a second ligation probe to a second target polynucleotide;

c. subjecting said first and second ligation probes to a ligation reaction in order to obtain one or more ligated products;

d. partitioning said one or more ligated products into two or more aqueous droplets within a continuous oil phase;

e. amplifying a sequence within said one or more ligated products to obtain amplified products;

f. determining a number of said two or more aqueous droplets that contain said amplified products; and

g. calculating a copy number of said first target polynucleotide.

41. The method of claim 40, wherein said target polynucleotide is not partitioned into said two or more aqueous droplets.

42. The method of claim 40, wherein during said amplifying of step (e) or during said determining of step (f), said two or more aqueous droplets remain substantially intact.

43. The method of claim 40, wherein said two or more aqueous droplets comprise on average more than one ligated probe and said method further comprises using an algorithm to calculate an average number of target ligated probes per aqueous droplet.

44. The method of claim 40, wherein said two or more aqueous droplets of step (d) comprise greater than 4,000 droplets.

45. The method of claim 44, wherein said greater than 4,000 droplets are combined together within a single chamber at a density of greater than 100,000 aqueous droplets/ml.

46. The method of claim 40, wherein said droplets are monodisperse droplets.

47. The method of claim 40, wherein said method can detect said first target polynucleotide within a population of genetic material comprising less than 1,000 copies of said first target polynucleotide

48. The method of claim 40, wherein said two or more aqueous droplets comprise on average more than one ligated probe and said method further comprises using an algorithm to calculate an average number of target ligated probes per aqueous droplet.

49. The method of claim 40, wherein each of said two or more aqueous droplets has, on average, a diameter of between 50 nm and 300 μ

m.

50. The method of claim 40, wherein said first target polynucleotide is a chromosomal segment associated with a genetic disorder.

51. The method of claim 40, wherein said ligation reaction ligates the 5′

region of said first ligation probe to the 3′

region of said first ligation probe.

52. The method of claim 40, wherein said first ligation probe is a padlock probe.

53. The method of claim 40, wherein said first ligation probe is a molecular inversion probe.

54. The method of claim 40, wherein said two or more aqueous droplets do not comprise a substantial number of beads conjugated to oligonucleotides.

55. The method of claim 40, wherein said continuous oil phase comprises an anionic fluorosurfactant.

56. The method of claim 55, wherein said anionic fluorosurfactant is an ammonium salt of an anionic fluorosurfactant Krytox™

.

57. The method of claim 56, wherein said Krytox is selected from a group consisting of Krytox AS, Krytox FSH, and morpholino derivative of Krytox FSH.

58. The method of claim 40, wherein said oil phase comprises a fluorinated oil.

59. The method of claim 40, wherein said two or more aqueous droplets do not comprise a microbead conjugated to an oligonucleotide.

60. A method of detecting a fetal genetic condition comprising:

a. obtaining a mixture of maternal and fetal genetic material comprising target polynucleotides;

b. combining said mixture with targeting oligonucleotides that bind said target polynucleotides;

c. subdividing said targeting oligonucleotides into reaction volumes, wherein at least one of said reaction volumes comprises no said target polynucleotide and no said targeting oligonucleotide;

d. performing an amplification reaction within said reaction volumes;

e. detecting a presence of said target polynucleotide or said targeting oligonucleotide within said reaction volumes; and

f. determining the relative level of said target polynucleotide in said mixture in order to detect a fetal genetic condition.

61. The method of claim 60, wherein said reaction volumes are aqueous droplets within a continuous oil phase.

62. The method of claim 60, wherein said targeting oligonucleotides comprise primer pairs.

63. The method of claim 60, wherein said targeting oligonucleotides comprise ligation probes.

64. The method of claim 60, wherein said targeting oligonucleotides comprise molecular inversion probes.

65. The method of claim 60, wherein said targeting oligonucleotides comprise padlock probes.

66. The method of claim 60, wherein said reaction volumes comprise, on average, greater than one copy of targeting oligonucleotide.

67. The method of claim 60, wherein said reaction volumes comprise, on average, greater than one copy of target polynucleotide.

68. The method of claim 60, wherein said reaction volumes further comprise primers to a reference polynucleotide.

69. The method of claim 60, wherein said reaction volumes further comprise a ligation probe to a reference polynucleotide.

70. The method of claim 63, wherein said ligation probes are amplified within said reaction volumes.

71. The method of claim 60, wherein said genetic material is not derived from a cellular sample that was selectively pre-enriched for fetal genetic material.

72. The method of claim 60, wherein said target polynucleotide is within a chromosome selected from the group consisting of chromosome 18, 13, 21, and X.

73. The method of claim 60, wherein said reaction volumes are aqueous droplets within an oil phase, said targeting oligonucleotides are ligation probes, and said determining in step (f) comprises comparing a number of droplets comprising an amplified product of said ligation probes with a number of droplets comprising an amplified product of ligation probes directed to a reference polynucleotide.

74. The method of claim 73, wherein said reference polynucleotide is a region of a chromosome that is not associated with a fetal genetic abnormality.

75. The method of claim 60, wherein said targeting oligonucleotides are ligation probes that become circular upon ligation following hybridization to a target polynucleotide.

76. A microcapsule comprising a ligated probe wherein said microcapsule is obtained by:

a. selectively binding a plurality of ligation probes to target polynucleotides within a genetic sample;

b. ligating a 5′

end of at least one of said bound ligation probes to a 3′

end of the same or different bound ligation probe, thereby obtaining at least one ligation product;

c. introducing an aqueous solution comprising said at least one ligation product into a device for generating droplets;

d. using said device to produce an aqueous droplet comprising said at least one ligation product, wherein said aqueous droplet is within an immiscible fluid; and

e. converting said droplet into a microcapsule comprising a solid-phase exterior.

77. The microcapsule of claim 76, wherein said converting comprises heating above 50°

C.

78. The microcapsule of claim 76, wherein said immiscible fluid comprises a fluorinated surfactant.

79. The microcapsule of claim 76, wherein said immiscible fluid comprises a fluorocarbon oil.

80. The microcapsule of claim 76, wherein said immiscible fluid comprises an anionic fluorosurfactant.

81. The microcapsule of claim 76, wherein said immiscible fluid comprises ammonium Krytox.

82. The microcapsule of claim 76, wherein said microcapsule does not comprise a bead bound to an oligonucleotide.

83. The microcapsule of claim 76, wherein said microcapsule remains substantially intact at temperatures above 70°

C.

84. The microcapsule of claim 76, wherein said ligation probes are capable of selectively binding to a target polynucleotide associated with a genetic disorder.

85. The microcapsule of claim 76, wherein said ligation probes are capable of selectively binding to a target polynucleotide associated with fetal aneuploidy.

86. The microcapsule of claim 76, wherein said genetic target is within a chromosome selected from the group consisting of chromosome 21, chromosome 13, chromosome 18, and the X chromosome.

87. The microcapsule of claim 76, wherein one or more of said ligation probes is a padlock probe.

88. The microcapsule of claim 76, wherein one or more of said ligation probes products is a circularized probe.

89. The microcapsule of claim 76, wherein one or more of said ligation probes is a molecular inversion probe.

90. A water-in-oil mixture comprising two or more aqueous droplets, wherein at least one of said two or more aqueous droplets comprises a first ligation probe directed to a first target polynucleotide and at least one of said two or more aqueous droplets comprises a second ligation probe directed to a second target polynucleotide.

91. The water-in-oil mixture of claim 90, wherein said first target polynucleotide and said second target polynucleotide are the same molecule.

92. The water-in-oil mixture of claim 90, wherein said first target polynucleotide has a different sequence than that of said second target polynucleotide.

93. The water-in-oil mixture of claim 90, wherein said first target polynucleotide has an identical sequence to said second target polynucleotide.

94. The water-in-oil mixture of claim 90, wherein said first target polynucleotide comprises a first region within a genomic segment and said second target polynucleotide comprises a second region within said genomic segment, wherein said first region does not have the same sequence as said second region.

95. The water-in-oil mixture of claim 90, wherein said water-in-oil mixture further comprises a ammonium krytox surfactant.

96. The water-in-oil mixture of claim 90, wherein said ammonium krytox surfactant is present in the oil phase of said mixture at a concentration of at least 0.01%.

97. The water-in-oil mixture of claim 90, wherein said water-in-oil mixture does not comprise amplified product of said first target nucleotide.

98. The water-in-oil mixture of claim 90, wherein said first ligation probe is a molecular inversion probe.

99. The water-in-oil mixture of claim 90, wherein said first ligation probe is a circularized probe.

100. The water-in-oil mixture of claim 90, wherein said first ligation probe is the linearized product of a circular probe that was subjected to enzymatic cleavage.

101. The water-in-oil mixture of claim 100, wherein said enzymatic cleavage is catalyzed by uracil-N-glycosylase or a restriction enzyme.