Method and device for identifying nucleotide, and method and device for determining nucleotide sequence of polynucleotide
First Claim
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1. A method for sequencing a polynucleotide, comprising:
- (a) providing a solid substrate comprising two electrodes that at least partially define a gap, wherein the two electrodes detect a current upon movement of the polynucleotide comprising a plurality of nucleotides through the gap;
(b) subjecting the polynucleotide to movement through the gap;
(c) measuring the current with time across the gap using the two electrodes during movement of the polynucleotide through the gap, wherein the current is measured at a sampling rate greater than about 1 kHz;
(d) determining durations and current levels associated with the current measured in (c) to identify individual nucleotides of the plurality of nucleotides with time, wherein the individual nucleotides are identified by comparing the current levels to a reference value(s); and
(e) determining a sequence of the polynucleotide based on an identification of the individual nucleotides of the plurality of nucleotides,wherein the gap is dimensioned such that the current is measured at the picoampere level under an applied voltage that is (i) greater than or equal to 0.25 V or (ii) less than or equal to 0.75 V.
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Abstract
The present invention provides technology that uses current measurements to identify nucleotides and determine a nucleotide sequence in polynucleotides. The present invention calculates a modal value of a tunnel current that arises when a nucleotide or polynucleotide for analysis passes through between electrodes, and then employs the calculated modal value. The present invention accordingly enables direct rapid implementation to identify nucleotides and to determine a nucleotide sequence in a polynucleotide without marking.
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Citations
30 Claims
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1. A method for sequencing a polynucleotide, comprising:
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(a) providing a solid substrate comprising two electrodes that at least partially define a gap, wherein the two electrodes detect a current upon movement of the polynucleotide comprising a plurality of nucleotides through the gap; (b) subjecting the polynucleotide to movement through the gap; (c) measuring the current with time across the gap using the two electrodes during movement of the polynucleotide through the gap, wherein the current is measured at a sampling rate greater than about 1 kHz; (d) determining durations and current levels associated with the current measured in (c) to identify individual nucleotides of the plurality of nucleotides with time, wherein the individual nucleotides are identified by comparing the current levels to a reference value(s); and (e) determining a sequence of the polynucleotide based on an identification of the individual nucleotides of the plurality of nucleotides, wherein the gap is dimensioned such that the current is measured at the picoampere level under an applied voltage that is (i) greater than or equal to 0.25 V or (ii) less than or equal to 0.75 V. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 11, 12)
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10. A method for sequencing a polynucleotide, comprising:
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(a) causing the polynucleotide to pass between electrodes, wherein the electrodes detect a current upon movement of the polynucleotide comprising a plurality of nucleotides between the electrodes; (b) using the electrodes to continuously measure current with time within a measurement time period; (c) identifying isolated current levels while continuously measuring the current with time, wherein at least a subset of the current levels are different upon comparison to a reference value(s); and (d) associating each of the isolated current levels with individual nucleotides of the plurality of nucleotides, thereby identifying a sequence of the polynucleotide, wherein a given nucleotide of the plurality of nucleotides is trapped between the electrodes for a specific duration while the current is continuously measured. - View Dependent Claims (13, 14, 15)
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16. A method for sequencing a polynucleotide, comprising:
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(a) causing the polynucleotide comprising a plurality of nucleotides to pass between electrodes separated by a distance comparable to a size of a given nucleotide of the plurality of nucleotides, wherein the electrodes detect a current upon movement of the polynucleotide between the electrodes; (b) measuring the current upon movement of the polynucleotide between the electrodes to yield a current distribution with time; (c) dividing the current distribution into a plurality of segments, wherein individual segments of the plurality of segments correspond to individual nucleotides of the plurality of nucleotides; and (d) comparing the plurality segments to one or more reference values to identify the individual nucleotides of the plurality of nucleotides, thereby generating a sequence of the polynucleotide; wherein in (b), the current is measured under an applied voltage that is (i) greater than or equal to 0.25 V or (ii) less than or equal to 0.75 V. - View Dependent Claims (17, 18, 19, 20, 21, 22, 23)
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24. A system for sequencing a polynucleotide, comprising:
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a solid substrate comprising two electrodes that at least partially define a gap, wherein the two electrodes detect a current upon movement of the polynucleotide comprising a plurality of nucleotides through the gap, and wherein the gap is in a break junction of the solid substrate; and a computing device that (i) measures the current with time across the gap using the two electrodes during movement of the polynucleotide through the gap, wherein the current is measured at a sampling rate greater than about 1 kHz, (ii) determines durations and current levels associated with the current to identify individual nucleotides of the plurality of nucleotides with time, and (iii) determines a sequence of the polynucleotide based on an identification of the individual nucleotides of the plurality of nucleotides, wherein the gap is dimensioned such that the current is measured at the picoampere level under an applied voltage that is (i) greater than or equal to 0.25 V or (ii) less than or equal to 0.75 V. - View Dependent Claims (25, 26, 27, 28, 29, 30)
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Specification