Inline-injection microdevice and microfabricated integrated DNA analysis system using same
First Claim
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1. A microfabricated structure for inline injection of a sample plug into a separation channel, comprising:
- a sample channel region for containing an unpurified sample of an analyte;
a capture channel region containing a capture matrix for forming a concentrated sample plug, wherein said capture matrix has a selective affinity for the analyte; and
a separation channel region to receive the sample plug and separate the analyte, wherein the capture channel and separation channel regions are contiguous and/or arranged in a line.
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Abstract
Methods and microfluidic circuitry for inline injection of nucleic acids for capillary electrophoresis analysis are provided. According to various embodiments, microfabricated structures including affinity-based capture matrixes inline with separation channels are provided. The affinity-based capture matrixes provide inline sample plug formation and injection into a capillary electrophoresis channel. Also provided are methods and apparatuses for a microbead-based inline injection system for DNA sequencing.
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Citations
45 Claims
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1. A microfabricated structure for inline injection of a sample plug into a separation channel, comprising:
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a sample channel region for containing an unpurified sample of an analyte; a capture channel region containing a capture matrix for forming a concentrated sample plug, wherein said capture matrix has a selective affinity for the analyte; and a separation channel region to receive the sample plug and separate the analyte, wherein the capture channel and separation channel regions are contiguous and/or arranged in a line. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15)
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16. A microfabricated structure for inline injection of a sample plug into a separation channel comprising:
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sample region means for containing an unpurified sample of an analyte; capture matrix means for forming a concentrated sample plug; means for inline injecting the sample plug into a separation channel; and the separation channel means for receiving the sample plug and separating the analyte.
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17. A microfabricated structure for paired-end sequencing comprising a plurality of sequencing elements, each element comprising:
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a thermal cycling reactor for producing forward and reverse extension fragments from a sequencing template; a forward capture channel region containing a forward capture matrix for concentrating forward extension fragments, wherein said forward capture matrix supports an oligonucleotide that selectively hybridizes to the forward extension fragments; a reverse capture channel region containing a reverse capture matrix for concentrating reverse extension fragments, wherein said reverse capture matrix supports an oligonucleotide that selectively hybridizes to the reverse extension fragments; a forward separation channel to separate the forward extension fragments, wherein the forward capture channel region and the forward separation channel region are contiguous and/or arranged in a line; and a reverse separation channel to separate the reverse extension fragments, wherein the reverse capture channel region and the reverse separation channel region are contiguous and/or arranged in a line. - View Dependent Claims (18, 19, 20, 21, 22)
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23. A microfabricated structure comprising:
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a distribution channel configured to distribute microreactor elements carrying multiple copies of a clonal sequencing template into each of a plurality of channels such that only one microreactor element will pass into one channel, wherein each channel comprises a thermal cycling chamber connected to a purification chamber connected to a component separation channel, wherein; thermal cycling extension fragments are produced from a microreactor element in the thermal cycling chambers; the purification chambers are configured to capture and concentrate the extension fragments; and the component separation are configured to analyze the extension fragments, and further wherein each component separation channel is contiguous with and/or is arranged in a line with a purification chamber and configured to receive the concentrated extension fragments via inline injection from that purification chamber.
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24. A system for performing sequencing comprising:
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means for shearing DNA into DNA fragments; means for ligating the DNA fragments to form a mixture of desired circular and contaminating linear products; means for exonuclease degradation for selectively removing the contaminating linear products; emulsion PCR reaction means for generating microspheres carrying multiple clonal copies of a single DNA sequencing template; fluorescent activated cell sorting (FACS) means for selecting which microspheres have a DNA sequencing template; microfluidic distribution channel means for distributing a selected microsphere with a DNA sequencing template into a thermal cycling chamber; valving means for ensuring that statistically only one microsphere will flow into one thermal cycling chamber; Sanger extension means, including the thermal cycling chambers, for producing thermal cycling extension fragments from the microspheres carrying multiple copies of the DNA sequencing template; capture means for capturing, purifying and concentrating the extension fragments into a sample plug; means for releasing the sample plug from the capture means; means for inline injecting the sample plug into a capillary array electrophoresis channel; and separation means, including a capillary array electrophoresis channel, for analyzing the extension fragments.
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25. A device comprising:
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a) a channel defining a flow path between a first end and a second end comprising; i. a capture channel region comprising an affinity capture matrix; ii. a separation channel region, wherein the capture channel region and the separation channel regions are contiguous and/or arranged in a line; b) a first electrode in electrical communication with a first end of the channel; c) a waste port in fluid communication with the capture channel region; d) a second electrode in electrical communication with the waste port, wherein a voltage applied between the first and second electrodes moves charged molecules through the capture channel region to the waste port; and e) a third electrode in electrical communication with a second end of the sample channel, wherein a voltage applied between the first and third electrodes moves charged molecules from the capture channel region through the separation channel region.
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26. A process of introducing an analyte in a sample to a separation channel comprising:
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introducing a sample containing an analyte to a sample channel region; driving the analyte in the sample to a capture channel region containing a capture matrix, said matrix having a selective affinity for the analyte; forming a concentrated sample plug in the capture channel region; and inline injecting the concentrated sample plug from the capture channel region into the separation channel. - View Dependent Claims (27, 28, 29, 30)
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31. A process for performing sequencing comprising:
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distributing microreactor elements with DNA sequencing templates into thermal cycling chambers, wherein each microreactor element has multiple clonal copies of a single unique sequencing template; producing thermal cycling extension fragments from the microreactor elements carrying multiple copies of a sequencing template; forming a concentrated sample plug of the extension fragments in a capture channel region comprising a capture matrix; inline injecting the sample plug from the capture matrix into a separation channel; and separating the extension fragments in the separation channel. - View Dependent Claims (32, 33, 34, 35, 36, 37, 38, 39)
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40. A process for performing sequencing comprising:
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distributing microreactor elements with DNA sequencing templates into thermal cycling chambers, wherein each microreactor element has multiple clonal copies of a single unique sequencing template; producing thermal cycling extension fragments from the microreactor elements carrying multiple copies of a sequencing template; forming a concentrated sample plug of the extension fragments in a capture channel region comprising a capture matrix; injecting the sample plug from the capture matrix into a separation channel, wherein at least about 50% of the extension fragments produced are injected into the separation channel. - View Dependent Claims (41, 42, 43)
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44. A method comprising:
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a) providing in a sample port a sample comprising analyte molecules and non-analyte molecules; b) moving the sample to a sample channel region; c) applying an electrical potential across a fluid path comprising the sample channel region, a capture channel region and a waste port, wherein the capture channel region comprises an affinity capture matrix configured to capture analyte molecules and wherein non-analyte molecules are moved by the electrical potential to the waste port; d) releasing captured analyte molecules from the affinity capture matrix; e) applying an electrical potential across a second fluid path comprising the capture channel region and a separation channel region of a separation channel, wherein the separation channel region is contiguous with and/or arranged in a line with the capture channel and wherein analyte molecules are moved by the electrical potential through the separation channel, whereby analyte molecules are resolved; and f) detecting the resolved analyte molecules in the separation channel. - View Dependent Claims (45)
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Specification