Systems and methods for automated reusable parallel biological reactions
First Claim
1. A method for sequencing, comprising:
- (a) providing a plurality of particles, wherein each particle of said plurality is coupled to on average at most one nucleic acid molecule;
(b) performing simultaneous nucleic acid amplification reactions on said nucleic acid molecules coupled to said particles, wherein said nucleic acid amplification reactions are performed while subjecting the particles to an electric field, thereby producing isolated, clonally-amplified nucleic acid molecules coupled to said plurality of particles;
(c) performing nucleic acid extension reactions on said clonally-amplified nucleic acid molecules using primers that hybridize to said clonally-amplified nucleic acid molecules; and
(d) detecting nucleic acid incorporation events from said extension reactions using a sensor array to determine a sequence of each of said clonally-amplified nucleic acid molecules, wherein each particle of said plurality of particles is adjacent to a sensor of said sensor array, and wherein each said sensor of said sensor array comprises two electrodes that are within a Debye length of their respective individual particle or within a Debye length of a nucleic acid molecule on said individual particle.
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Abstract
A method comprises magnetically holding a bead carrying biological material (e.g., nucleic acid, which may be in the form of DNA fragments or amplified DNA) in a specific location of a substrate, and applying an electric field local to the bead to isolate the biological material or products or byproducts of reactions of the biological material. For example, the bead is isolated from other beads having associated biological material. The electric field in various embodiments concentrates reagents for an amplification or sequencing reaction, and/or concentrates and isolates detectable reaction by-products. For example, by isolating nucleic acids around individual beads, the electric field can allow for clonal amplification, as an alternative to emulsion PCR. In other embodiments, the electric field isolates a nanosensor proximate to the bead, to facilitate detection of at least one of local pH change, local conductivity change, local charge concentration change and local heat. The beads may be trapped in the form of an array of localized magnetic field regions.
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Citations
18 Claims
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1. A method for sequencing, comprising:
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(a) providing a plurality of particles, wherein each particle of said plurality is coupled to on average at most one nucleic acid molecule; (b) performing simultaneous nucleic acid amplification reactions on said nucleic acid molecules coupled to said particles, wherein said nucleic acid amplification reactions are performed while subjecting the particles to an electric field, thereby producing isolated, clonally-amplified nucleic acid molecules coupled to said plurality of particles; (c) performing nucleic acid extension reactions on said clonally-amplified nucleic acid molecules using primers that hybridize to said clonally-amplified nucleic acid molecules; and (d) detecting nucleic acid incorporation events from said extension reactions using a sensor array to determine a sequence of each of said clonally-amplified nucleic acid molecules, wherein each particle of said plurality of particles is adjacent to a sensor of said sensor array, and wherein each said sensor of said sensor array comprises two electrodes that are within a Debye length of their respective individual particle or within a Debye length of a nucleic acid molecule on said individual particle. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 14, 15)
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10. A method for nucleic acid sequencing, comprising:
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(a) directing a plurality of particles onto an array of sensors, wherein an individual particle comprises a nucleic acid molecule, and wherein an individual sensor of said array comprises a pair of electrodes that are electrically coupled to a Debye length of said individual particle during nucleic acid sequencing; (b) positioning said individual particle at said individual sensor; (c) directing a plurality of primers onto said array; (d) performing a primer extension reaction at said individual sensor; and (e) during or subsequent to performing said primer extension reaction, measuring a change in impedance of said individual particle with the aid of said pair of electrodes that are each electrically coupled to said Debye length of said individual particle. - View Dependent Claims (11, 12, 13, 16, 17, 18)
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Specification