PARTICLE-ASSISTED NUCLEIC ACID SEQUENCING

• US 20170183715A1
• Filed: 02/08/2017
• Published: 06/29/2017
• Est. Priority Date: 12/19/2008
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Abate, Adam R., WEITZ, DAVID A.
• Granted Patent: US 10,457,977 B2
• Time in Patent Office: Days
• 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

1-83. -83. (canceled)

84. A method of sequencing a target nucleic acid, the method comprising:

passing a microfluidic droplet containing a target nucleic acid through a microfluidic channel;

exposing the target nucleic acid to a plurality of particles, at least some of which have nucleic acid probes fastened thereto, wherein at least some of the particles are contained within said microfluidic droplet containing the target nucleic acid and said nucleic acid probes comprises a nucleic acid probe that can hybridize with said target nucleic acid, and wherein the particles comprise a gel;

hybridizing said nucleic acid probes to the target nucleic acid; and

at least partially determining the sequence of the target nucleic acid.

85. The method of claim 84, wherein at least one of the particles is associated with at least one identification entity.

86. The method of claim 85, wherein said at least partially determining the sequence of the target nucleic acid comprises determining the at least one identification entity.

87. The method of claim 85, further comprising determining the at least one identification entity.

88. The method of claim 87, wherein the at least one identification entity is determined using fluorescence.

89. The method of claim 84, wherein at least some of the nucleic acid probes contain at least six residues.

90. The method of claim 84, wherein at least some of the nucleic acid probes contain DNA.

91. The method of claim 84, wherein the target nucleic acid comprises DNA.

92. The method of claim 84, wherein the target nucleic acid comprises a signaling entity.

93. The method of claim 84, further comprising determining association of the target nucleic acid and at least some of the particles.

94. The method of claim 93, wherein determining the association of the target nucleic acid and at least some of the particles comprises determining a signaling entity.

95. The method of claim 94, wherein the signaling entity is not covalently bound to the target nucleic acid.

96. The method of claim 84, further comprising amplifying the target nucleic acid.

97. The method of claim 84, wherein at least one of the nucleic acid probes is immobilized relative to at least one identification entity.

98. The method of claim 97, wherein the at least one identification entity comprises at least one fluorophore.

99. The method of claim 84, further comprising determining association of at least some of the nucleic acid probes and the target nucleic acid contained within the microfluidic droplet.

100. The method of claim 84, wherein the plurality of particles comprise at least 200 different nucleic acid probe sequences.

101. The method of claim 84, wherein the plurality of particles comprise at least 1000 different nucleic acid probe sequences.

102. The method of claim 84, wherein said at least partially determining the sequence of the target nucleic acid comprises sequencing by hybridization.

103. The method of claim 84, wherein said at least partially determining the sequence of the target nucleic acid comprises determining a hybridization spectrum.