DEVICES AND METHODS FOR ANALYZING BIOMOLECULES AND PROBES BOUND THERETO
First Claim
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1. A method for mapping a target biomolecule, the method comprising the steps of:
- a) providing a single-stranded or double-stranded target biomolecule;
b) providing an apparatus comprising first and second fluid chambers in fluid communication with one another, wherein the first and second fluid chambers are separated by a structure defining a member selected from the group consisting of a nanopore, a microchannel, and a nanochannel, and wherein the apparatus comprises at least one pair of electrodes defining at least one detector volume within the structure;
c) providing at least one probe set, said probe set comprising a first plurality of identical probes that selectively hybridize to complementary regions on the target biomolecule;
d) hybridizing the probes to the target biomolecule to provide a partially hybridized biomolecule having probes hybridized to complementary regions thereon;
e) coating at least a portion of the partially hybridized biomolecule with one or more proteins;
f) translocating the partially hybridized biomolecule through the at least one detector volume;
g) monitoring, as a function of time, changes in an electrical property detected by the at least one pair of electrodes defining the at least one detector volume as the partially hybridized biomolecule translocates therethrough; and
h) differentiating between hybridized and non-hybridized regions of the target biomolecule based at least in part on the detected changes in the electrical property in the at least one detector volume, thereby mapping at least a portion of the target biomolecule.
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Abstract
Devices and methods for sequencing biomolecules include improving signal-to-noise ratio of detection of relative positions of probes hybridized to a biomolecule by coating at least a portion of the biomolecule with a protein prior to its translocation through a structure defining a nanopore, microchannel or nanochannel.
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Citations
75 Claims
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1. A method for mapping a target biomolecule, the method comprising the steps of:
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a) providing a single-stranded or double-stranded target biomolecule; b) providing an apparatus comprising first and second fluid chambers in fluid communication with one another, wherein the first and second fluid chambers are separated by a structure defining a member selected from the group consisting of a nanopore, a microchannel, and a nanochannel, and wherein the apparatus comprises at least one pair of electrodes defining at least one detector volume within the structure; c) providing at least one probe set, said probe set comprising a first plurality of identical probes that selectively hybridize to complementary regions on the target biomolecule; d) hybridizing the probes to the target biomolecule to provide a partially hybridized biomolecule having probes hybridized to complementary regions thereon; e) coating at least a portion of the partially hybridized biomolecule with one or more proteins; f) translocating the partially hybridized biomolecule through the at least one detector volume; g) monitoring, as a function of time, changes in an electrical property detected by the at least one pair of electrodes defining the at least one detector volume as the partially hybridized biomolecule translocates therethrough; and h) differentiating between hybridized and non-hybridized regions of the target biomolecule based at least in part on the detected changes in the electrical property in the at least one detector volume, thereby mapping at least a portion of the target biomolecule. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)
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30. A method for sequencing a target biomolecule, the method comprising the steps of:
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a) providing a single-stranded or a double-stranded target biomolecule; b) providing an apparatus comprising first and second fluid chambers in fluid communication with one another, wherein the first and second fluid chambers are separated by a structure defining a member selected from the group consisting of a nanopore, a microchannel, and a nanochannel, and wherein the apparatus comprises at least one pair of electrodes defining at least one detector volume within the structure; c) providing at least two probe sets, each of said probe sets comprising a plurality of identical probes that selectively hybridize to complementary regions on the target biomolecule, each probe set hybridizing to a different complementary region, wherein at least one region complementary to the first probe set shares a subregion that is complementary to the second probe set; d) hybridizing the first probe set to a first sample of the target biomolecule to provide a first partially hybridized biomolecule sample having first probes hybridized to complementary regions thereon; e) at least partially coating the first partially hybridized biomolecule sample with one or more proteins; f) translocating the first partially hybridized biomolecule sample through the detector volume; g) monitoring, as a function of time, changes in an electrical property detected by the at least one pair of electrodes defining the at least one detector volume as the first partially hybridized biomolecule sample translocates therethrough; h) differentiating between hybridized and non-hybridized regions of the first target biomolecule sample based at least in part on the detected changes in the electrical property in the at least one detector volume; i) hybridizing the second probe set to a second sample of the target biomolecule to provide a second partially hybridized biomolecule sample having second probes hybridized to complementary regions thereon; j) at least partially coating the second partially hybridized biomolecule sample with one or more proteins; k) translocating the second partially hybridized biomolecule sample through the detector volume; l) monitoring, as a function of time, changes in an electrical property detected by the at least one pair of electrodes defining the at least one detector volume as the second partially hybridized biomolecule sample translocates therethrough; m) differentiating between hybridized and non-hybridized portions of the second target biomolecule sample based at least in part on the detected changes in the electrical property in the at least one detector volume; and n) assembling correlated data sets from the first target biomolecule sample and the second target biomolecule sample, thereby sequencing at least a portion of the target biomolecule. - View Dependent Claims (31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58)
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59. An apparatus for analyzing a target biomolecule, the apparatus comprising:
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a) first and second fluid chambers in fluid communication with one another, wherein the first and second fluid chambers are separated by a structure defining at least one nanopore; at least one pair of electrodes positioned on opposite sides of the structure and defining a detector volume therethrough, the electrodes being in communication with an electrical signal detector and data collection device for respectively detecting and recording changes in an electrical property as the target biomolecule translocates through the detector volume; and c) a driving force generator for translocating the target biomolecule from the first fluid chamber to the second fluid chamber through the detector volume. - View Dependent Claims (60, 61, 62, 63, 64)
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65. An apparatus for analyzing a target biomolecule, the apparatus comprising:
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a) first and second fluid chambers in fluid communication with one another, wherein the first and second fluid chambers are separated by a structure defining a member selected from the group consisting of a nanochannel and a microchannel; b) at least one pair of electrodes laterally offset from one another along the channel and defining at least one detector volume therein, the electrodes being in communication with an electrical signal detector and data collection device for respectively detecting and recording changes in an electrical property as the target biomolecule translocates through the at least one detector volume; and c) a driving force generator for translocating the target biomolecule from the first fluid chamber to the second fluid chamber through the at least one detector volume. - View Dependent Claims (66, 67, 68, 69, 70, 71, 72, 73, 74, 75)
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Specification