Apparatus and methods for sequencing nucleic acids in microfluidic systems
First Claim
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1. A microfluidic apparatus for determining relative positions in a target nucleic acid sequence that are occupied by a first nucleotide, comprising:
- a microscale separation channel having first and second ends for separating nucleic acid fragments by size;
a first source containing a nested set of first nucleotide termination fragments of the target nucleic acid in fluid communication with the separation channel, wherein the fragments terminate at each of the positions in the target occupied by the first nucleotide;
a second source containing a nested set of second nucleotide termination fragments of the target nucleic acid, in fluid communication with the separation channel, wherein the fragments terminate at each of the positions occupied by the second nucleotide;
a means for mixing a first concentration of the first nested set with a second concentration of the second nested set in a first mixture, the first concentration being detectably different from the second concentration;
a means for injecting a portion of the first mixture into the separation channel and transporting the portion through the separation channel; and
a means for detecting separate fragments of the first and second nested sets in the separation channel.
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Abstract
The present invention is generally directed to microfluidic systems and methods of using such systems in the determination of the nucleotide sequence of target nucleic acid sequences (referred to herein as the "target"). In particular, the present invention provides methods and systems for determining the relative positions within a target nucleic acid sequence that are occupied by a given nucleotide, e.g., A, T, G or C, by separating mixtures of nested sets of fragments of the target nucleic acid, which sets each include fragments that terminate in a different given nucleotide.
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28 Claims
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1. A microfluidic apparatus for determining relative positions in a target nucleic acid sequence that are occupied by a first nucleotide, comprising:
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a microscale separation channel having first and second ends for separating nucleic acid fragments by size; a first source containing a nested set of first nucleotide termination fragments of the target nucleic acid in fluid communication with the separation channel, wherein the fragments terminate at each of the positions in the target occupied by the first nucleotide; a second source containing a nested set of second nucleotide termination fragments of the target nucleic acid, in fluid communication with the separation channel, wherein the fragments terminate at each of the positions occupied by the second nucleotide; a means for mixing a first concentration of the first nested set with a second concentration of the second nested set in a first mixture, the first concentration being detectably different from the second concentration; a means for injecting a portion of the first mixture into the separation channel and transporting the portion through the separation channel; and a means for detecting separate fragments of the first and second nested sets in the separation channel. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)
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19. A microfluidic system for determining relative positions in a target nucleic acid sequence that are occupied by a given nucleotide, comprising:
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a body structure comprising a top portion, a bottom portion and an interior portion; a microscale separation channel disposed in the interior portion, the separation channel having first and second ends; an injection channel disposed in the interior portion and intersecting the separation channel at a first intersection; first and second reservoirs disposed in the body structure, each of the first and second reservoirs being in fluid communication with the injection channel on a first side of the intersection; wherein the first reservoir contains a first nested set of fragments of the target nucleic acid sequence each of the fragments in the first nested set terminating at a different position occupied by the given nucleotide, and the second reservoir contains a second nested set of fragments of the target nucleic acid sequence, each of the fragments in the second nested set terminating in a nucleotide different from the given nucleotide; an electrical controller operably coupled to each of the first and second reservoirs and the first and second ends, for concomitantly transporting a first concentration of the first nested set and a second concentration of the second nested set through the intersection the first and second nested sets forming a first mixture, injecting a portion of the first mixture into the separation channel and transporting the portion along the separation channel.
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20. A method of determining positions in a target nucleic acid sequence occupied by a first nucleotide, comprising:
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providing a microfluidic device that comprises; a separation channel for separating nucleic acid sequences by size; a first source containing a nested set of first nucleotide termination fragments of the target nucleic acid in fluid communication with the separation channel, wherein the fragments terminate at each of the positions in the target occupied by the first nucleotide; a second source containing a nested set of second nucleotide termination fragments of the target nucleic acid, in fluid communication with the separation channel, wherein the fragments terminate at each of the positions occupied by the second nucleotide; mixing a first concentration of the first nested set . . . the first concentration being detectablY different from the second concentration; transporting a portion of the first mixture through the separation channel to separate the fragment in each of the first and second nested sets; detecting fragments separated in the transporting seep and distinguishing fragments from the first nested set from fragments from the second nested set based upon their relative concentration; determining the positions within the target nucleic acid sequence occupied by the first nucleotide by comparing the size of fragments in the first nested set to the size of fragments in the second nested set. - View Dependent Claims (21, 22, 23, 24, 25, 26, 27)
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28. A method of determining positions in a target nucleic acid sequence occupied by a given nucleotide, comprising:
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providing a microfluidic device, the device comprising; at least a first separation channel having at least first and second ends; at least first injection channel intersecting the separation channel at a first intersection; at least first and second reservoirs in fluid communication with the injection channel; placing a first nested set of fragments of the nucleic acid sequence in the first reservoir, each fragment in the nested set terminating at a different position occupied by the given nucleotide; placing a second nested set of fragments of the target nucleic acid in the second reservoir, each fragment in the second nested set terminating in a nucleotide different from the given nucleotide; concomitantly flowing a first concentration of the first nested set and a second concentration of the second nested set, into the injection channel to form a first mixture, the second concentration being determinably different from the first concentration; injecting a portion of the first mixture into the separation channel whereupon each of the fragments in each of the first and second nested sets are separated by size; distinguishing the fragments of the first nested set from the fragments of the second nested set by their relative concentration; and determining positions occupied by the given nucleotide based upon the size of the fragments in the first nested set relative to the size of the fragments in the second nested set.
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Specification