PARTICLE-ASSISTED NUCLEIC ACID SEQUENCING

• US 20140303039A1
• Filed: 04/28/2014
• Published: 10/09/2014
• Est. Priority Date: 12/19/2008
• Status: Abandoned Application

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Weitz, David A., Abate, Adam R.
• Application Number:
  US14/262,895
• Publication Number:
  US 20140303039A1
• Time in Patent Office: Days
• US Class Current: 506/16
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• 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 2525/204 specific length of the oligonucleotides
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

This invention generally relates to particle-assisted nucleic acid sequencing. In some embodiments, sequencing may be performed in a microfluidic device, which can offer desirable properties, for example, minimal use of reagents, facile scale-up, and/or high throughput. In one embodiment, a target nucleic acid may be exposed to particles having nucleic acid probes. By determining the binding of the particles to the target nucleic acid, the sequence of the target nucleic acid (or at least a portion of the target nucleic acid) can be determined. The target nucleic acid may be encapsulated within a fluidic droplet with the particles having nucleic acid probes, in certain instances. In some cases, the sequence of the target nucleic acid may be determined, based on binding of the particles, using sequencing by hybridization (SBH) algorithms or other known techniques.

Claims

1-60. -60. (canceled)

61. A composition, comprising:

a plurality of particles, at least some of which have nucleic acid probes fastened thereto, wherein the plurality of particles contains at least 120 unique nucleic acid probe sequences, such that each of the particles contains only one of the nucleic acid probe sequences and is associated with an identification entity.

62. The composition of claim 61, wherein the plurality of particles is contained within a liquid.

63. The composition of claim 61, wherein the plurality of particles is contained within a microfluidic device.

64. The composition of claim 61, wherein at least some of the plurality of particles are contained within a fluidic droplet.

65. The composition of claim 61, wherein the at least 120 unique nucleic acid probes consists essentially of 6-mers.

66. The composition of claim 61, wherein the identification entity comprises at least one fluorescent molecule.

67. The composition of claim 61, wherein the plurality of particles contains at least 200 unique nucleic acid probe sequences.

68. The composition of claim 61, wherein the plurality of particles contains at least 1000 unique nucleic acid probe sequences.

69. The composition of claim 61, wherein the plurality of particles contains at least 4000 unique nucleic acid probe sequences.

70. A composition comprising:

a plurality of particles, at least some of which have nucleic acid probes fastened thereto, wherein the plurality of particles contains at least one unique nucleic acid probe sequence, the nucleic acid probe having a signaling entity attached thereto, such that each of the particles contains only one of the at least one nucleic acid probe sequences and is associated with an identification entity.

71. The composition of claim 70, wherein the plurality of particles contains at least two unique nucleic acid probe sequences attached to the same particle.

72. The composition of claim 71, wherein at least one of the signaling entities attached to the at least two unique nucleic acid probes sequences comprises an enhancer or quencher.

73. The composition of claim 70, wherein the at least one unique nucleic acid probe sequence is a 6-mer.

74. The composition of claim 70, wherein the identification entity comprises at least one fluorescent molecule.

75. The composition of claim 70, wherein the plurality of particles contains at least 2 unique nucleic acid probe sequences.

76. The composition of claim 70, wherein the plurality of particles contains at least 10 unique nucleic acid probe sequences.

77. The composition of claim 70, wherein the plurality of particles contains at least 120 unique nucleic acid probe sequences.

78. A kit, comprising:

a plurality of particles, at least some of which have nucleic acid probes fastened thereto, wherein the plurality of particles contains at least 120 unique nucleic acid probe sequences, such that each of the particles contains only one of the nucleic acid probe sequences and is associated with an identification entity.

79. The kit of claim 78, wherein the plurality of particles is contained within a liquid.

80. The kit of claim 78, wherein the plurality of particles is contained within a microfluidic device.

81. The kit of claim 78, wherein at least some of the plurality of particles are contained within a fluidic droplet.

82. The kit of claim 78, wherein the at least 120 unique nucleic acid probes consists essentially of 6-mers.

83. The kit of claim 78, wherein the identification entity comprises at least one fluorescent molecule.