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US 2013/0096013 A1Published application
Methods and systems for electronic sequencing
AssigneeGenapsys, Inc.
InventorsHesaam Esfandyarpour, Mark Oldham, Eric Nordman
Life of this patent
Estimating the term…
Abstract
The present invention provides for methods and systems for Electronic DNA sequencing, single molecule DNA sequencing, and combinations of the above, providing low cost and convenient sequencing.
Claims48
1-12. (canceled)
A method for sequencing DNA comprising:
- a) contacting DNA proximate to nanosensors in a nanosensor array with a fluid containing one or more of sequencing reagents, primers, polymerase and nucleotides, wherein the nucleotides are modified nucleotides with a charge modifying structure;
- b) sequencing said DNA by detecting a nucleotide incorporation event with the nanosensors, the nanosensors detecting a change in associated electrical, chemical, or thermal signals;
- c) washing said nanosensor array to remove at least one of sequencing reagents, primers, polymerase, nucleotides, and DNA;
- and
- d) repeating steps a, b, c, and d to obtain the sequence information.
2 more elements
A method for reverse sequencing of DNA comprising:
- a) contacting DNA proximate to nanosensors in a nanosensor array with one or more of sequencing reagents, primers, polymerase and pools of nucleotides, wherein the polymerase has 3′ to 5′ exonuclease activity and the nucleotide pools are missing at least one nucleotide;
- b) sequencing said DNA by detecting a nucleotide incorporation event with the nanosensors, the nanosensors detecting a change in associated electrical, chemical, or thermal signals;
- c) washing said nanosensor array to remove at least one of sequencing reagents, primers, polymerase, pools of nucleotides, and DNA;
- and
- d) repeating steps a, b, c, and d to obtain the sequence information.
2 more elements
A method for sequencing DNA using ligation comprising:
- a) contacting DNA proximate to nanosensors in a nanosensor array with one or more of sequencing reagents, primers, DNA ligase and pools of probe oligonucleotides;
- b) sequencing said DNA by detecting a nucleotide incorporation event with the nanosensors, the nanosensors detecting a change in associated electrical, chemical, or thermal signals;
- c) washing said nanosensor array to remove at least one of sequencing reagents, primers, ligase, pools of probe oligonucleotides, and DNA;
- and
- d) repeating steps a, b, c, and d to obtain the sequence information.
2 more elements
A method for polynucleotide sequencing, comprising:
- a) circularizing a DNA template and immobilizing the DNA template proximate to a sensor,
- b) contacting the DNA template with a sequencing primer for extension, one or more nucleotide triphosphates for incorporation, and a strand-displacing polymerase under conditions suitable for extending the sequencing primer through the DNA template multiple times, and
- c) detecting the sequence of nucleotide incorporation by sensing a change in associated electrical, chemical, or thermal signals.
A method for sequencing and re-sequencing one or more polynucleotides comprising:
- a) providing a hairpin primer with a restriction site on a portion of the polynucleotide;
- and
- b) sequencing the polynucleotide by using a nanosensor to sense a change in associated electrical, chemical, or thermal signals.
A method for sequencing polynucleotides, comprising:
- contacting sequencing reaction components with a nanosensor etched under so that both sides of the nanosensor are accessible to sense a change in electrical, chemical, or thermal signals associated with a nucleotide incorporation event.
A method of sequencing a single polynucleotide molecule using a nanosensor in a nanosensor array, comprising attaching a polymerase enzyme to the nanosensor, binding the polynucleotide molecule and associated primers to said polymerase enzyme, and measuring a change in associated electrical, chemical, or thermal signals upon incorporation of a nucleotide to the polynucleotide molecule.
A method for preparing DNA for sequencing comprising:
- a) performing blunt end ligation on a DNA molecule with ligands that have different binding reagents on the 3′ and 5′ end of said ligands;
- b) subsequently binding said DNA molecule to electrodes of a nanosensor;
- c) contacting the DNA molecule with a sequencing primer for extension, one or more nucleotide triphosphates for incorporation, and a strand-displacing polymerase under conditions suitable for extending the sequencing primer through the DNA template multiple times;
- and
- d) detecting the sequence of nucleotide incorporation by sensing a change in associated electrical, chemical, or thermal signals.
2 more elements
An apparatus comprising:
- a) a base layer;
- b) a plurality of nanosensors in an array for binding with polynucleotides in a fluid sample, each nanosensor having a structure with conductors exposed to said fluid sample comprising:
- c) a first oxide layer on the substrate;
- d) a first conductive layer on the first oxide layer;
- e) a second oxide layer on the first conductive layer;
- f) a second conductive layer on the second oxide layer;
- g) a third oxide layer on the second conductive layer;
- h) a plurality of array channels for flowing fluid sample in and out of said array, wherein the nanosensors are in proximity to said channel and said channel is optionally etched under the structure;
- and
- i) a detection means coupled to each nanosensor and configured and arranged to sense a change in associated electrical, chemical, or thermal signals.
7 more elements
An apparatus comprising:
- a) a base layer;
- b) a plurality of nanosensors in an array for binding with polynucleotides in a fluid sample, each nanosensor having a structure with coaxial conductors exposed to said fluid sample comprising:
- i. a first oxide layer in the substrate;
- ii. a first conductive layer located in the substrate, in contact with the first oxide layer;
- iii. a second conductive layer located in the substrate in contact with the first oxide layer;
- iv. an oxide layer optionally covering a portion of the first or second conductive layers that are exposed to said fluid sample;
- v. a plurality of array channels for flowing fluid sample in and out of said array, wherein the nanosensors are in proximity to said channel;
- and
- vi. a detection means coupled to each nanosensor and configured and arranged to sense a change in associated electrical, chemical, or thermal signals.
6 more elements
A system comprising:
- a) a nanosensor array with associated reaction chambers configured for at least one of clonal amplification and sequencing of polynucleotides, optionally configured for both clonal amplification and sequencing in a sequential manner in the same reaction chamber;
- b) DNA proximate to the nanosensors and in contact with one or more of sequencing reagents, primers, DNA polymerase and nucleotides;
- and
- c) wherein the nanosensors are configured to detect a nucleotide incorporation event by detecting a change in associated electrical, chemical, or thermal signals.
1 more element
Dependencies are inferred from the claim text.